Carboxymethylated cross-linked tetrameric hemoglobin

ABSTRACT

The present invention includes compositions containing carboxymethylated cross-linked hemoglobin where the cysteine moiety of the hemoglobin includes thiol protecting group and where the hemoglobin is incapable of binding with the nitric oxide. Preferably, the hemoglobin is deoxygenated, endotoxin free, and stroma free. The present invention also includes method of preparation, process of preparation and the method of supplementing blood in mammals.

BACKGROUND OF THE INVENTION

One of the limitations on the use of blood in an emergency setting is a requirement to type and cross-match the blood to minimize the risk of transfusion rejection. Saline cross-matching may require at least 10 minutes and a complete type and cross-match can take up to an hour. Furthermore, the risk of HIV transmission has been estimated to be 1 in 500,000 units of blood and the risk of hepatitis C transmission has been estimated to be 1 in 3,000 units. The safety of blood supply is an issue in developing countries, where the risk of infectious disease transmission is relatively higher. Up to 25% of the blood is discarded in developing countries because of the presence of infectious disease. Hence, there are pressing factors to find a potential blood substitute that avoids the disease transmission and provides rapid response to improve chances of survival.

The examples of two “blood substitute” categories are volume expanders and oxygen therapeutics. Volume expanders are inert and merely increase blood volume. Oxygen therapeutics mimics human blood's oxygen transport ability. Oxygen therapeutics can be divided in two categories based on transport mechanism: perfluorocarbon based, and hemoglobin (protein) based. In hemoglobin-based products, pure hemoglobin separated from red blood cells may not be useful since it may cause renal toxicity and it may have unsuitable oxygen transport characteristics when separated from red blood cells.

Some of the characteristics of a blood substitute are: virus-free, non-toxic, non-immunogenic, satisfactory oxygen carrying capacity and circulatory persistence to permit effective oxygenation of tissues, long shelf life, storage at room temperature, avoids disease transmission, no requirement of blood typing, and deployable for use by first responders such as, paramedics, military medics on front lines etc. These characteristics provide a rapid response to blood loss and can improve chances of survival.

The present invention disclosed herein provides compositions and methods for deoxygenated, endotoxin free, stroma free, carboxymethylated cross-linked tetrameric hemoglobin.

SUMMARY OF THE INVENTION

The present invention relates to a proteinaceous iron containing compound having a molecular weight of about 60,000 daltons to about 500,000 and having at least one cysteine moiety wherein the cysteine moiety includes a thiol protecting group such that the proteinaceous compound is incapable of binding nitric oxide at the cysteine site. Preferably, the proteinaceous iron containing compound is a cross-linked tetrameric hemoglobin, and more preferably compound the non-pyrogenic, endotoxin free, oxygen free and stroma free and of low viscosity. In some embodiments, the proteinaceous compound has been cross linked with bis 3′,5′ dibromo salicyl fumarate. In some embodiments, the hemoglobin has been modified by reaction with pyridoxal-5′-phosphate. In some embodiments, the hemoglobin is human hemoglobin. In some preferred embodiments, the hemoglobin is bovine or porcine hemoglobin. In some embodiments, the thiol protecting group is selected from the group consisting of 4-pyridylmethyl, acetylaminomethyl, alkoxyalkyl, triphenylmethyl, carboxymethyl, acetyl, benzyl, benzoyl, tert-butoxycarbonyl, p-hydroxyphenacyl, p-acetoxybenzyl, p-methoxybenzyl, 2,4-dinitrophenyl, isobutoxymethyl, tetrahydropyranyl, acetamidomethyl, benzamidomethyl, bis-carboethoxyethyl, 2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, N-alkyl carbamate, and N-alkoxyalkyl carbamate. In some embodiments, the thiol protecting group is a carboxymethyl group. In some embodiments, the proteinaceous iron containing compound increases oxygen carrying capacity. In some preferred embodiments, the proteinaceous iron containing compound transports oxygen with a p50 of about 20 to about 45. Some embodiments relate to a composition comprising the proteinaceous iron containing compound and a pharmaceutically acceptable carrier. Some embodiments relate to a container containing a composition comprising the proteinaceous iron containing compound of the aforementioned aspect of the invention.

Another aspect of the invention relates to a method of supplementing blood of a mammal comprising administering to the mammal a composition comprising a proteinaceous iron containing compound having a molecular weight of about 60,000 daltons to about 500,000 and having at least one cysteine moiety wherein the cysteine moiety includes a thiol protecting group such that the proteinaceous compound is incapable of binding nitric oxide at the cysteine site, and a pharmaceutically acceptable carrier. In some preferred embodiments, the proteinaceous iron containing compound is a cross-linked tetrameric hemoglobin. In some preferred embodiments, the hemoglobin is non-pyrogenic, endotoxin free, oxygen free and stroma free. In some embodiments, the hemoglobin has been crosslinked with bis 3′,5′dibromo salicyl fumarate. In some embodiments, the hemoglobin has been modified by reaction with pyridoxal-5′-phosphate. In some embodiments, the hemoglobin is human hemoglobin. In some preferred embodiments, the hemoglobin is bovine or porcine hemoglobin. In some embodiments, the thiol protecting group is selected from the group consisting of 4-pyridylmethyl, acetylaminomethyl, alkoxyalkyl, triphenylmethyl, carboxymethyl, acetyl, benzyl, benzoyl, tert-butoxycarbonyl, p-hydroxyphenacyl, p-acetoxybenzyl, p-methoxybenzyl, 2,4-dinitrophenyl, isobutoxymethyl, tetrahydropyranyl, acetamidomethyl, benzamidomethyl, bis-carboethoxyethyl, 2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, N-alkyl carbamate, and N-alkoxyalkyl carbamate. In some embodiments, the thiol protecting group is a carboxymethyl group. In some embodiments, the proteinaceous iron containing compound increases oxygen carrying capacity. In some preferred embodiments, the proteinaceous iron containing compound transports oxygen with a p50 of about 20 to about 40. In some embodiments, the administering is by an implant, injection or transfusion. In some embodiments, the mammal suffers from anemia, anemia related conditions, hypoxia or ischemia. In some embodiments, the mammal needs blood transfusion. In some embodiments, the mammal is in trauma.

Yet another aspect of the invention relates to a process for a preparation of a cross linked tetrameric hemoglobin wherein the hemoglobin has a cysteine group attached to a thiol protecting group comprising: (a) removing endotoxin from a preparation containing red blood cells; (b) lysing the red blood cells; (c) separating hemoglobin by removing stroma from the lysed red blood cells; (d) removing oxygen from the hemoglobin; (e) adding to a hemoglobin solution a reagent which provides a thiol protecting group for a cysteine of the hemoglobin, and (f) separating a hemoglobin which has a cysteine group attached to a thiol protecting group. In some embodiments, the reagent is selected from the group consisting of 4-pyridylmethyl chloride, alkoxyalkylchloride, dimethoxymethane, N-(hydroxymethyl)acetamide, triphenylmethyl chloride, acetyl chloride, acetic anhydride, haloacetamide, iodoacetate, benzyl chloride, benzoyl chloride, di-tert-butyl dicarbonate, p-hydroxyphenacyl bromide, p-actoxybenzyl chloride, p-methoxybenzyl chloride, 2,4-dinitrophenyl fluoride, tetrahydropyran, acetamidohydroxymethane, acetone, bis-carboethoxyethene, 2,2,2-trichloroethoxycarbonyl chloride, tert-butoxycarbonyl chloride, alkyl isocyanate, and alkoxyalkyl isocyanate. In some preferred embodiments, the haloacetamide is iodoacetamide. In some embodiments, the thiol protecting group is selected from the group consisting of 4-pyridylmethyl, acetylaminomethyl, alkoxyalkyl, triphenylmethyl, carboxymethyl, acetyl, benzyl, benzoyl, tert-butoxycarbonyl, p-hydroxyphenacyl, p-acetoxybenzyl, p-methoxybenzyl, 2,4-dinitrophenyl, isobutoxymethyl, tetrahydropyranyl, acetamidomethyl, benzamidomethyl, bis-carboethoxyethyl, 2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, N-alkyl carbamate, and N-alkoxyalkyl carbamate. In some preferred embodiments, the thiol protecting group is a carboxymethyl.

In some embodiments of the aforementioned aspect of the invention, the process further comprises: (a) removing oxygen from the hemoglobin which has a cysteine group attached to a thiol protecting group; and cross linking the hemoglobin which has a cysteine group attached to a thiol protecting group. In some preferred embodiments, the oxygen is removed by contactor membrane technology. In some preferred embodiments, the cross linked tetrameric hemoglobin is non-pyrogenic, endotoxin free, oxygen free and stroma free. In some embodiments, the hemoglobin which has a cysteine group attached to a thiol protecting group is crosslinked with bis 3′,5′ dibromo salicyl fumarate. In some embodiments, the hemoglobin which has a cysteine group attached to a thiol protecting group is modified by reaction with pyridoxal-5′-phosphate. In some embodiments, the red blood cells are human red blood cells. In some preferred embodiments, the red blood cells are bovine or porcine red blood cells. In some embodiments, the cross linked tetrameric hemoglobin is incapable of binding to nitric oxide. In some embodiments, the cross linked tetrameric hemoglobin increases oxygen carrying capacity. In some preferred embodiments, the cross linked tetrameric hemoglobin transports oxygen with a p50 of about 20 to about 45.

Yet another aspect of the invention relates to a method for producing a hemoglobin wherein a cysteine in the hemoglobin is attached to a thiol protecting group, by a process comprising: (a) adding to a hemoglobin solution a reagent which provides a thiol protecting group for a cysteine of the hemoglobin, and (b) separating a hemoglobin which has a cysteine group attached to a thiol protecting group.

In another aspect of the invention, a hemoglobin of the invention is a thiol blocked stroma free hemoglobin (TBSFH) that may be safely stored for extended periods. This TBSFH may be a stable intermediate which can endure packaging, shipping and further handling to yield another hemoglobin of the invention.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 is a flow chart showing the steps of the methods as disclosed herein.

FIG. 2 depicts the time course of a lysis experiment showing resistance of WBC lysis.

FIG. 3 depicts the size standards used in electrophoretic separations as disclosed within.

FIG. 4A depicts electrophoretic separations of native hemoglobin, dXCMSFH, and size standard in the range around 20 KDa.

FIG. 4B depicts the electrophoretic separation of native hemoglobin, dXCMSFH, and size standards for the full chromatographic runs.

FIG. 5 shows oxygen affinity curves for bovine whole blood, stroma free Hb, cross linked hemoglobin, and fresh human blood.

FIG. 6 depicts the HPLC separation of products of the cross-linking reaction:

FIGS. 7A-D depict the cardiac output, systemic vascular resistance, and mean arterial pressure, respectively, in the pig safety trial.

DETAILED DESCRIPTION OF THE INVENTION

The term “endotoxin free” or its grammatical equivalents as used herein, means a hemoglobin that has been treated to reduce exposure to and to remove substantially or completely all endotoxin as measured by a very sensitive assay technique. Thus, endotoxin-free hemoglobin can have less than or the equivalent of the amount of endotoxin present in Water for Injection (WFI).

The term “non-pyrogenic” or its grammatical equivalents as used herein, means a hemoglobin that may be administered to a mammal without causing a febrile reaction.

The term “oxygen free”, “deoxygenated”, or its grammatical equivalents as used herein, means a hemoglobin that has been treated to remove substantially or completely all oxygen. Oxygen-free hemoglobin thus is substantially or completely in the higher energy “tense” or “T” configuration.

The term “stroma free” or its grammatical equivalents as used herein, means a hemoglobin that has been treated to remove substantially or completely all stromal material, such that the preparation no longer exhibits the immunoreactivity to blood type antigens characteristic of RBCs. Stroma-free hemoglobin thus substantially or completely lacks the toxic and/or pyrogenic properties associated with preparations of hemolyzed red blood cells, and thus can be administered to an individual without causing toxicity or inflammatory reaction. In addition it is substantially free of membrane lipids and cellular carbonic anhydrase.

The term “dXCMSFH” refers to the deoxygenated endotoxin-free, stroma-free, thiol blocked, preferably carboxymethylated cross-linked hemoglobin of the present invention.

The term “dTBSFH” refers to the deoxygenated endotoxin-free, stroma-free, thiol-blocked uncross-linked hemoglobin of the present invention.

The term “dCMSFH” refers to the deoxygenated endotoxin-free, -stroma free, carboxymethylated uncross-linked hemoglobin of the invention and is a specific example of a dTBSFH.

The present invention relates to a carboxymethylated, cross-linked tetrameric hemoglobin wherein at least one cysteine moiety in the hemoglobin has been protected with a thiol (or sulfhydryl) protecting group such that the thiol group in the cysteine moiety is not available for binding with nitric oxide (NO). Preferably, at least two cysteine moieties in the hemoglobin are protected with a thiol (or sulfhydryl) protecting group such that the thiol group in the cysteine moiety is not available for binding with nitric oxide (NO). More preferably, the hemoglobin is non-pyrogenic, endotoxin free, oxygen free, and stroma free. Bovine hemoglobin contains only two thiol groups (Cys 93 on the beta chain) which are involved in binding NO. Hence preferably, both thiol groups are protected with a thiol protecting group in bovine Hb. Human hemoglobin contains six thiol groups, two of which (Cys 93 on the beta chain) are involved in binding NO. Preferably these two thiols are protected and up to six thiol groups may be protected with a thiol protecting group in human Hb.

The protection of cysteine in hemoglobin can avoid binding of NO at the thiol group in the cysteine moiety. The protected cysteine containing hemoglobin can prevent vasoactivity of the blood vessels. NO is a smooth-muscle relaxant that functions via activation of guanylate cyclase and the production of cGMP or by direct activation of calcium-dependent potassium channels. The increase in the free Hb can result in an increase in the NO binding. The increase in the NO binding can result in transient or sustained changes in hemodynamic properties of blood responding to vasoactive substances or the lack of vasoactive regulatory substances and may lead to hypertension. It has been demonstrated that NO may bind to the reactive sulfhydryls of Hb and may be transported to and from the tissues in a manner analogous to the transport of oxygen by heme groups (Jia et al., Nature 1996; 80:221-226). Protection of thiol or sulfhydryl groups in the hemoglobin molecule may prevent the binding of NO to the hemoglobin at the thiol site and hence prevent acute vasoactive response of the blood vessels causing a hypertensive reaction.

Hemoglobin Compositions

Hemoglobin Molecule

Hemoglobin or haemoglobin (Hb) is a proteinaceous iron-containing compound having a molecular weight of about 60,000 daltons which transports oxygen in the red blood cells of the blood in mammals and other animals. Hemoglobin transports oxygen from the lungs to the rest of the body, such as to the muscles, wherein it releases part of the oxygen load. The hemoglobin molecule is an assembly of four globular protein subunits. Each subunit is composed of a protein chain tightly associated with a non-protein heme group. Each individual protein chain arranges in a set of α-helix structural segments connected together in a “myoglobin fold” arrangement, so called because this arrangement is the same folding motif used in the heme/globin proteins. This folding pattern contains a pocket which is suitable to strongly bind the heme group. A heme group consists of an iron atom held in a heterocyclic ring, known as a porphyrin. This iron atom is the site of oxygen binding. The iron atom is bonded equally to all four nitrogens in the center of the ring, which lie in one plane. Two additional bonds perpendicular to the plane on each side can be formed with the iron to form the fifth and sixth positions, one connected strongly to the protein, the other available for binding of oxygen. The iron atom can either be in the Fe²⁺ or Fe³⁺ state, but ferrihaemoglobin (methemoglobin) (Fe³⁺) cannot bind oxygen.

In adult humans, the hemoglobin type is a tetramer (which contains 4 subunit proteins) called hemoglobin A, consisting of two α and two β subunits non-covalently bound, each made of 141 and 146 amino acid residues, respectively. This is denoted as α₂β₂. The subunits are structurally similar and about the same size. Each subunit has a molecular weight of about 16,000 daltons, for a total molecular weight of the tetramer of about 64,000 daltons. The four polypeptide chains are bound to each other by salt bridges, hydrogen bonds and hydrophobic interactions. There are two kinds of contacts between the α and the β chains: α₁β₁ and α₁β₂. However, adult hemoglobin may also comprise δ globin subunits. The δ globin subunit replaces β globin and pairs with α globin as α₂β₂ to form hemoglobin A2.

Natural hemoglobin is composed of a tetrameric arrangement of α and β polypeptide chains. Within the RBC, the association of the α chain with its corresponding β chain is very strong and does not disassociate under physiological conditions. The association of one α/β dimer with another α/β dimer, however, is fairly weak and outside of the RBC, the two dimers may disassociate even under physiological conditions. Upon disassociation, the dimer is filtered through the glomerulus. The rapid clearing of stroma free hemoglobin (SFH) by the kidney is a consequence of its quaternary molecular arrangement.

To avoid such removal, hemoglobin can be conjugated or cross-linked by various methods known in the art. One of the methods is by conjugating Hb to another molecule such as polyethylene glycol (PEG), which forms a hydrophilic shield around the Hb molecule and simultaneously increases its size which in turn increases its circulatory half-life. Hb can also be cross-linked intramolecularly to prevent dissociation of the tetramer into αβ dimers and/or cross-linked intermolecularly to form polymers which also increases the oxygen carrier's size and thus increases its circulatory half-life. Using site-specific cross-linking reagents, intramolecular covalent bonds may be formed, which may convert Hb into a stable tetramer, thus preventing its dissociation into αβ dimers. On the other hand, the use of non-specific cross-linkers such as glutaraldehyde may lead to non-specific covalent bonding between amino acid residues residing within and between Hb tetramers. This leads to the formation of hemoglobin polymers (polyHb) of various molecular weights. Chemical reagents with multi-aldehyde functionalities can be used as cross-linking agents. These include molecules such as glutaraldehyde, ring-opened raffinose and dextran. In the case of aldehydes, the formation of covalent cross-links may be initiated by the carbonyl group of the aldehyde reacting with an amino group present in the Hb tetramer. Polymerization of Hb into larger molecules may increase the intravascular half-life of the polyHb with respect to tetrameric Hb and prevent Hb dissociation into αβ dimers. PolyHb may be eventually filtered out of the systemic circulation throughout the kidneys, the lymphatic system, and the reticuloendothelial system (RES).

Several other chemical agents can be used to cross-link hemoglobin α/β dimers and prevent their filtration by the glomerulus into the urine, and yet maintain the oxygen transport properties of native hemoglobin. Bis 3′,5′dibromo salicyl fumarate (BDBF) is an activated diester of fumaric acid that has been used as a cross-linker to cross-link hemoglobin (Tye, U.S. Pat. No. 4,529,719, hereby incorporated by reference in its entirety). Bis 3′,5′dibromo salicyl fumarate effects this change by associating the salicyl moieties with the sites known to bind aspirin within hemoglobin, and then effecting crosslinking by the fumarate active functionalities with the alpha and beta chains. This maintains the two dimers in proper orientation for cross-linking with lysine residues. A slight molar excess of BDBF cross-linker to hemoglobin (1.2:1.0), under sub-optimum conditions, may yield 70% cross-linked hemoglobin molecules. Cross-linking the α or β chains of hemoglobin can prevent disassociation of the tetramer.

Bovine Hb is structurally similar to human Hb. The bovine Hb contains two α chains and two β chains. The two sulfhydryl groups in the bovine Hb are β Cys 93, while the sulfhydryls in human Hb are found at α Cys 104, β Cys 93 and β Cys 112. Bovine Hb and human Hb differ in the way in which oxygen affinity is affected. The affinity for oxygen by human Hb is affected by the binding of 2,3-diphosphoglycerate (2,3-DPG) into the 2,3-DPG pocket of human Hb, while bovine Hb, possessing a further internal salt bridge, has its affinity for oxygen affected by the ionic strength of the local environment. An advantage to affinity modulation by ionic strength versus that induced by 2,3-DPG binding is that sufficient concentration of ionic species is generally present in plasma while 2,3-DPG is only contained within RBCs. Thus, the oxygen affinity of acellular bovine Hb can be modulated more easily than acellular human Hb. This advantage of modulation of affinity by general ionic interaction can be built back into human Hb by reacting it with pyridoxal-5-phosphate (PLP). PLP modifies human Hb by introducing a negative charge near a penultimate β chain histidine residue and by removing a positive charge at the amino terminal end of the same chain, An altered human Hb of this class can now respond more similarly to bovine Hb to charged species in the local environment and not solely depend on binding of 2,3-DPG to affect oxygen affinity.

Hemoglobin as Oxygen Transporter

The compositions and methods of the present invention relate to a carboxymethylated, cross-linked tetrameric hemoglobin where a at least one cysteine moiety in the hemoglobin molecule includes a thiol protecting group, for example, a carboxymethyl group such that the hemoglobin has a oxygen carrying capacity with a p50 of about 20 to about 45 mm Hg. Preferably, the hemoglobin is non-pyrogenic, endotoxin free, oxygen free, and stroma free. The p50 of stroma free human hemoglobin in solution can be approximately 12 to 17 mm Hg as compared to native, RBC associated hemoglobin p50 of approximately 27 mm Hg. The hemoglobin of the present invention can be a p50 of about 20 to about 45 mm Hg. The p50 test can be done in vitro in the absence of CO₂. Therefore, carboxymethylated cross-linked hemoglobin of the present invention has high oxygen carrying capacity and is functionally superior to native hemoglobin.

When hemoglobin binds oxygen, it shifts from the high energy “tense” or “T” state (deoxygenated or oxygen free) to the lower energy “relaxed” or “R” state (oxygenated). Human α and β globin genes have been cloned and sequenced (Liebhaber et al., Proc. Natl. Acad. Sci. (U.S.A). 77:7054-58 (1980); Marotta et al., J. Biol. Chem. 252:5040-43 (1977); and Lawn et al., Cell 21:647 (1980), all of which are incorporated by reference in their entirety). The tetrameric structure of T state deoxyhemoglobin has increased stability from six ionic bonds and while in the T state, hemoglobin is effectively prevented from disassociating into dimers. In this conformation, the beta cleft contact area between the two beta chains (also known as the beta pocket, phosphate pocket, and 2,3-diphosphoglycerate binding site) in deoxyhemoglobin is substantially different than in oxyhemoglobin. The changed conformation of the beta cleft in the T state is believed to explain the decreased oxygen affinity stabilized by 2,3-diphosphoglycerate. The T state of hemoglobin is stable and resistant to denaturation. Thus, cross-linking the SFH addresses both the issues of oxygen affinity and the problem of rapid filtration by the kidney.

In the tetrameric form of normal adult hemoglobin, the binding of oxygen is a cooperative process. The binding affinity of hemoglobin for oxygen is increased by the oxygen saturation of the molecule. As a consequence, the oxygen binding curve of hemoglobin is sigmoidal, or S-shaped. This positive cooperative binding may be achieved through steric conformational changes of the hemoglobin protein complex. When one subunit protein in hemoglobin becomes oxygenated, it induces a conformational or structural change in the whole complex causing the other subunits to gain an increased affinity for oxygen.

Hemoglobin's oxygen-binding capacity may be decreased in the presence of carbon monoxide because both gases compete for the same binding sites on hemoglobin, carbon monoxide binding preferentially to oxygen. Carbon dioxide occupies a different binding site on the hemoglobin. Hemoglobin can bind protons and carbon dioxide which causes a conformational change in the protein and facilitates the release of oxygen. Protons bind at various places along the protein and carbon dioxide binds at the α-amino group forming carbamate. Conversely, when the carbon dioxide levels in the blood decrease (i.e., around the lungs), carbon dioxide is released, increasing the oxygen affinity of the protein. This control of hemoglobin's affinity for oxygen by the binding and release of carbon dioxide is known as the Bohr effect.

The binding of oxygen is affected by molecules such as carbon monoxide (CO) (for example from tobacco smoking, cars and furnaces). CO competes with oxygen at the heme binding site. Hemoglobin binding affinity for CO is 200 times greater than its affinity for oxygen, meaning that small amounts of CO may reduce hemoglobin's ability to transport oxygen. When hemoglobin combines with CO, it forms a very bright red compound called carboxyhemoglobin. When inspired air contains CO levels as low as 0.02%, headache and nausea may occur; if the CO concentration is increased to 0.1%, unconsciousness may follow. In heavy smokers, up to 20% of the oxygen-active sites can be blocked by CO. Hemoglobin also has competitive binding affinity for sulfur monoxide (SO), nitrogen dioxide (NO₂), nitric oxide, and hydrogen sulfide (H₂S). The iron atom in the heme group is in the Fe²⁺ oxidation state to support oxygen transport. Oxidation to Fe³⁺ state converts hemoglobin into methemoglobin, which cannot bind oxygen. Nitrogen dioxide and nitrous oxide are capable of converting hemoglobin to methemoglobin.

The tetrameric structure of human hemoglobin provides a binding site for 2,3-diphosphoglycerate. Inside red blood cells, the binding of 2,3-diphosphoglycerate to hemoglobin decreases the hemoglobin's oxygen affinity to a level compatible with oxygen transport. The binding of 2,3-diphosphoglycerate to hemoglobin is weak and may require high concentrations (i.e., concentrations approaching 1M or more) in order to modify the affinity of hemoglobin for oxygen. Thus, when the red blood cells are ruptured to produce stroma free hemoglobin (SFH), the 2,3-diphosphoglycerate may not be retained in close proximity to the hemoglobin and may disassociate from the hemoglobin. As a consequence, unless further modified, SFH may exhibit a higher affinity for oxygen than does hemoglobin in RBCs. The increased affinity of the SFH for oxygen may be quite significant since, under physiological conditions, it may be unable to release the bound oxygen to the tissues. Bovine hemoglobin does not require 2,3-DPG to maintain a p50 for oxygen in the range of 30 mm. In people acclimated to high altitudes, the concentration of 2,3-diphosphoglycerate (2,3-DPG) in the blood is increased, which allows these individuals to deliver a larger amount of oxygen to tissues under conditions of lower oxygen tension.

A variant hemoglobin, called fetal hemoglobin (HbF, α₂γ₂), is found in the developing fetus, and binds oxygen with greater affinity than adult hemoglobin. This means that the oxygen binding curve for fetal hemoglobin is left-shifted (i.e., a higher percentage of hemoglobin has oxygen bound to it at lower oxygen tension), in comparison to that of adult hemoglobin. As a result, fetal blood in the placenta is able to take oxygen from maternal blood.

Hemoglobin in the Presence of Nitric Oxide (NO)

Unprotected thiol on the cysteine moiety of the hemoglobin may bind with NO and the trapping of NO may cause vasoconstriction of blood vessels which may lead to high blood pressure or hypertension. Protection of the thiol site at the cysteine moiety of the hemoglobin prevents the binding of NO to the hemoglobin at the thiol site thereby preventing acute vasoconstrictive activity of blood vessels and hypertension response.

Nitric oxide is a regulator of the arterial perfusion of any tissue. Nitric oxide is synthesized and released by the endothelium in the arterial wall, where it can be bound by hemoglobin in red blood cells. When a tissue is receiving too much oxygen, nitric oxide is not released and the arterial wall muscle contracts making the vessel diameter smaller, thus decreasing perfusion. When demand for oxygen increases, the endothelium releases nitric oxide, which causes vasodilatation. The nitric oxide control of arterial perfusion operates over the distance that NO diffuses after release from the endothelium. Nitric oxide is also needed to mediate certain inflammatory responses. For example, nitric oxide produced by the endothelium inhibits platelet aggregation. Consequently, as nitric oxide is bound by cell-free hemoglobin, platelet aggregation may be increased. As platelets aggregate, they release potent vasoconstrictor compounds such as thromboxane A₂ and serotonin. These compounds may act synergistically with the reduced nitric oxide levels caused by hemoglobin scavenging resulting in significant vasoconstriction. In addition to inhibiting platelet aggregation, nitric oxide also inhibits neutrophil attachment to cell walls, which in turn may lead to cell wall damage. Because nitric oxide binds to hemoglobin inside the red blood cell, it is expected that the nitric oxide may bind to the free Hb (stroma free cross linked tetrameric Hb) as well.

The ability of Hb solutions to affect blood pressure has been known. It has been demonstrated that cross-linked Hb solutions could increase mean arterial pressure as much as 25-30% in a dose-dependent manner within 15 min of administration and that the effect could last as long as 5 h. The effects are believed to be due largely to the trapping of NO by Hb (Katsuyama et al., Artif Cells Blood Substit Immobil Biotechnol 1994; 22: 1-7; Schultz et al., J Lab Clin Med 1993; 122:301-308, hereby incorporated by reference in its entirety). NO is a smooth-muscle relaxant that functions via activation of guanylate cyclase and the production of cGMP or by direct activation of calcium-dependent potassium channels. Therefore, the increased binding of NO, which could result from increased free Hb could result in transient or sustained changes in hydrodynamic properties of blood or vasoactive substances and may lead to hypertension. It has been demonstrated that NO may bind to the reactive sulfhydryls of Hb and may be transported to and from the tissues in a manner analogous to the transport of oxygen by heme groups (Jia et al., Nature 1996; 80:221-226).

It has been observed that free Hb infusion causes vasoconstriction of the blood vessels, resulting in extremely high blood pressures in many affected areas. This may make the affected blood vessels very porous, and the free Hb solution can leak into the surrounding tissues causing the tissues to turn purple. In rabbit models, transfusion of free Hb through the ear vein has caused cerebral vasculature ischemia and death. Therefore, it is important to minimize the impact of administration of free Hb on the arterial system during administration. Vasoactive agents such as verapamil, atenocard, sildenafil citrate, etc., may be administered to the patient prior to free Hb infusion. This is intended to ensure that the arterial system is minimally changed during infusion. Nitric oxide and verapamil are preferred vasoactive agents. Slow channel calcium blockers (or a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), such as sildenafil citrate) may also be helpful in the prevention of the severe vasoconstriction. However, a slower infusion rate may not be preferred with respect to a trauma patient.

Method for Producing Carboxymethylated Cross-Linked Hemoglobin

One aspect of the present invention relates to a process for a preparation of a carboxymethylated, cross-linked tetrameric hemoglobin. Yet another aspect of the present invention includes a method of producing a carboxymethylated, cross-linked tetrameric hemoglobin. Preferably, the hemoglobin is a non-pyrogenic, endotoxin free, oxygen free, and stroma free.

The steps for some of the embodiments of the present invention are depicted in FIG. 1. Without limiting the scope of the present invention, the steps can be performed independent of each other or one after the other. One or more steps can be skipped in the methods of the present invention. The method of producing the hemoglobin of the present invention can involve step 101 comprising removing endotoxin from a preparation containing red blood cells; step 102 comprising lysing the red blood cells; step 103 comprising separating hemoglobin by removing stroma, including membranes and leucocytes, from the lysed red blood cells; step 104 comprising removing oxygen from the hemoglobin; step 105 comprising adding to a hemoglobin solution a reagent which provides a thiol protecting group for a cysteine of the hemoglobin; step 106 comprising of separating the hemoglobin which has a cysteine group attached to a thiol protecting group; step 107 comprising cross linking the hemoglobin; and step 108 comprising preparing a non-pyrogenic, endotoxin free, oxygen free, stroma free, cysteine protected, crosslinked hemoglobin. Without limiting the scope of the present invention, the order of the steps may be changed depending on the requirement/s for producing a hemoglobin according to this invention.

A. Sources of Hemoglobin

Blood, RBCs or hemoglobin used in the present invention may be obtained from a variety of mammalian sources, such as, for example, human, bovine (genus bos), bison (genus bison), ovine (genus ovis), porcine (genus sus) sources, other vertebrates or transgenically-produced hemoglobin. Alternatively, the stroma-free hemoglobin used in the present invention may be synthetically produced by a bacterial, or more preferably, by a yeast, mammalian cell, or insect cell expression vector system (Hoffman, S. J. et al., U.S. Pat. No. 5,028,588 and Hoffman, et al., WO 90/13645, both herein incorporated by reference). Alternatively, hemoglobin can be obtained from transgenic animals; such animals can be engineered to express non-endogenous hemoglobin (Logan, J. S. et al. PCT Application No. PCT/US92/05000; Townes, T. M. et al., PCT Application No. PCT/US/09624, both herein incorporated by reference in their entirety). Preferably, the stroma-free hemoglobin used in the present invention is isolated from bison, bovine or human sources.

The genus bos includes, subgenus bos including bos taurus (western cattle, including oxen and aurochs) and bos aegyptiacus; subgenus bibos including bos frontalis (gaur, gayal or Indian bison) and bos javanicus (banteng); subgenus novibos including bos sauveli (kouprey or grey ox), and; subgenus poephagus including bos grunniens (yak; also bos mutus). The bos taurus, includes similar types from Africa and Asia such as, bos indicus, the zebu; and the bos primigenius, the aurochs. The bos gurus includes subspecies, bos gaurus laosiensis, bos gaurus gaurus (such as in India, Nepal) also called “Indian bison”, bos gaurus readei, bos gaurus hubbacki (such as in Thailand, Malaysia), and bos gaurus frontalis, a domestic gaur, or a gaur-cattle hybrid breed.

Bison is a taxonomic genus containing six species within the subfamily bovinae. The bison may be called buffalo in Asia (such as water buffalo) and Africa (such as African buffalo). The genus bison includes species such as, bison latifrons (long-horned bison), bison antiquus, bison occidentalis, bison priscus, bison bison, bison bison bison, bison bison athabascae, bison bonasus, bison bonasus bonasus, bison bonasus caucasicus, and bison bonasus hungarorum. In some embodiments of the present invention, the hemoglobin is from genus bos or bison. In some preferred embodiments of the present invention, the hemoglobin is from genus bison. It shall be understood that any mammalian species may be used as a source of hemoglobin and is within the scope of the present invention.

Bovine Hb is easier to obtain and more abundant than human Hb. Typically human Hb extracted from outdated RBCs is used for Hb-based artificial blood substitute research. However, outdated RBCs are not available in sufficient quantities to produce large amounts of viable artificial blood substitute.

Hemoglobin, whether derived from an animal, synthetic or recombinant, may be composed of the “naturally existing” hemoglobin protein, or may contain some or be entirely composed of, a mutant hemoglobin protein. Preferred mutant hemoglobin proteins include those whose mutations result in more desirable oxygen binding/release characteristics. Examples of such mutant hemoglobin proteins include those provided by Hoffman, S. L. et al. (U.S. Pat. Nos. 5,028,588 and 5,776,890) and Anderson, D. C. et al. (U.S. Pat. Nos. 5,844,090 and 5,599,907), all herein incorporated by reference in their entirety.

The blood can be collected from live or freshly slaughtered donors. It is preferred that the blood be collected in a sanitary manner. At or soon after collection, the blood is typically mixed with at least one anticoagulant to prevent significant clotting of the blood. Suitable anticoagulants for blood are as classically known in the art and include, for example, sodium citrate, ethylenediaminetetraacetic acid and heparin. When mixed with blood, the anticoagulant may be in a solid form, such as a powder, or in an aqueous solution. It is understood that the blood solution source can be from a freshly collected sample or from an old sample, such as expired human blood from a blood bank. Further, the blood solution could previously have been maintained in frozen and/or liquid state. It is preferred that the blood solution is not frozen prior to use in this method.

Prior to introducing the blood solution to anticoagulants, antibiotic levels in the blood solution, such as penicillin, may be assayed. Antibiotic levels may be determined to provide a degree of assurance that the blood sample is not burdened with an infecting organism by verifying that the donor of the blood sample was not being treated with an antibiotic. Alternatively, a herd management program to monitor and insure the lack of disease in or antibiotic presence from treatment of the cattle may be used. The blood solution may be strained prior to or during the anticoagulation step, for example by straining, to remove large aggregates and particles. A 150 micron filter is a suitable strainer for this operation.

B. Cleansing of Membranes and Equipment

Any of a variety of assays may be employed to demonstrate the non-pyrogenicity of the compositions of the present invention, for example, but are not limited to, interleukin-6 and other cytokine induction (Pool, E. J. et al., J. Immunoassay 19:95-111 (1998), and; Poole, S. et al., Dev. Biol. Stand. 69:121-123 (1988)); human monocytoid cell line assays (Eperon, S. et al., J. Immunol. Meth. 207:135-145 (1997), and; Taktak, Y. S. et al., J. Pharm. Pharmacol. 43:578-582 (1991)); the limulus amoebocyte lysate (LAL) test (Fujiwara, H. et al., Yakugaku Zasshi 110:332-40 (1990), and; Martel F. et al., Rev Fr Transfus Immunohematol 28:237-250 (1985)) and the rabbit pyrogen test (Bleeker W. K. et al., Prog Clin Biol Res 189:293-303 (1985); Simon, S. et al., Dev. Biol. Stand. 34:75-84 (1977), and; Allison, E. S. et al., Clin. Sci. Mol. Med. 45:449-458 (1973)), all references incorporated herein in their entirety. The rabbit pyrogen test is the preferred pyrogenicity assay. It is understood that other methods of removing the pyrogen are known in the art and are within the scope of the present invention.

C. Eliminating or Removing Endotoxins and Lysing RBC

Serum lipases, such as lipase A, do not inactivate endotoxins bound to the hemoglobin molecule. Therefore, endotoxins remain active toxins when taken up by the hepatocyte metabolizing the hemoglobin. Friedman, H. I. et al. reported triad hepatoxicity in a rat model consistent with this theory (See, Friedman, H. I. et al., Lab Invest 39:167-77 (1978), and; Colpan et al., U.S. Pat. No. 5,747,663) have reported a process for reducing or removing endotoxins from a cellular lysate solution. Wainwright et al. (U.S. Pat. No. 5,627,266) have described an endotoxin binding protein immobilized to a solid support and the use of this molecule in the removal of endotoxins from solution.

In some embodiments of the present invention, the elimination of contamination with endotoxins can be ensured by preventing the addition of endotoxins to the chemical processes of the present invention. Typically, endotoxins are added inadvertently by using endotoxin contaminated water or the simple process of bacteria exposure during collection. Measurement of endotoxins can be difficult, and standard LAL binding assays do not work in the presence of hemoglobin since initial collection endotoxin binds strongly to hemoglobin. Water and the blood collection can be the most likely candidates for introduction of endotoxins since increased number of steps in the preparation of hemoglobin may increase the level of toxicity. Preparations using dialysis and filtration methods can expose the hemoglobin to a thousand volumes of water/buffer that may be contaminated with endotoxin. Membrane systems may be pretreated with NaOH or NaOCl to reduce or eliminate endotoxins. These materials may then be flushed and cleaned from the various devices.

It is preferred that all membranes, and equipment used to produce the hemoglobin of the present invention be cleansed in a manner sufficient to cause the removal or elimination of endotoxin that may be present on such materials and equipment. Preferably, such cleansing is accomplished by pre-washing surfaces and equipment that may come into contact with the hemoglobin of the present invention using a dilute solution of hemoglobin. Such a solution serves to bind endotoxin and hence to remove residual endotoxin that may be present on such membranes or equipment. See, for example, Tye, U.S. Pat. No. 6,894,150. The dilute solution of hemoglobin is preferably discarded after each use.

The RBCs in the blood solution can be washed by suitable means, such as by diafiltration or by a combination of discrete dilution and concentration steps with at least one solution, such as an isotonic solution, to separate RBCs from extracellular plasma proteins, such as serum albumins or antibodies (e.g., immunoglobulins (IgG)). It is understood that the RBCs can be washed in a batch or continuous feed mode. Acceptable isotonic solutions are well known in the art and include solutions, such as, for example, citrate/saline solution or PBS, having a pH and osmolarity which does not rupture the cell membranes of RBCs and which displaces the plasma portion of the whole blood. Sources of purified water which can be used in the method of invention includes distilled water, deionized water, water-for-injection (WFI) and/or low pyrogen water (LPW). WFI, which is preferred, is deionized, distilled water. The specific method of purifying water is not as important as the requirement that it needs to be low in endotoxin content.

The water and the reagents used in the present invention are substantially free from endotoxin contamination. Preferably, the water and the reagents used in the present invention are completely free from endotoxin contamination. One way to reduce the risk of endotoxin contamination can be to reduce the amount of water and reagent buffers exposed to the hemoglobin preparation. Therefore, under some embodiments of the present invention, the hemoglobin preparations are made using counter-flow or counter-current dialysis for equilibration of buffers and/or removal of reaction products. Counter flow dialysis methods are suitable for use in the present invention are commercially available e.g., VariPerm M, bitop, Witten (see, e.g., Schwarz, T. et al, Electrophoresis 15:1118-1119 (1994)), Spectrum Laboratories, Inc., Laguna Hills, Calif., etc. It is estimated that the hollow fiber technique may yield a hemoglobin preparation of the present invention that has a 100 fold reduction in the amount of endotoxin as compared to standard synthesis techniques. It is understood that other methods of removing the endotoxins are known in the art and are within the scope of the present invention.

It is understood that methods generally known in the art for separating RBCs from other blood components can be employed. For example, sedimentation, wherein the separation method does not rupture the cell membranes of a significant amount of the RBCs, such as less than about 30% of the RBCs, prior to RBC separation from the other blood components such as, white blood cells and platelets. Hemoglobin may be released from the erythrocyte by hypotonic lysis in deionized water. Preferably, lysis is done in five to twenty volumes of deionized water. More referably, lysis is done in twenty volumes of deionized water. RBCs are collected in a flow process where after the addition of saline, RBCs are allowed to lyse but just before the WBCs begin to lyse, an approximate additional 10% volume of a 9% saline solution is added to arrive at a total concentration of 0.9% saline content overall. This timed increase in salinity prevents WBCs from lysing. The stroma and WBCs are removed from the lysed RBCs by filteration. Lysis means can use various lysis methods, such as mechanical lysis, chemical lysis, hypotonic lysis or other known lysis methods which release hemoglobin without significantly damaging the ability of the Hb to transport and release oxygen. Other methods of erythrocyte lysis, such as “slow hypotonic lysis” or “freeze thaw”, may also be employed. See, e.g., Chan et al., J. Cell Physiol. 85:47-57 (1975), incorporated by reference in its entirety. In some embodiments of the present invention, the cells are lysed by flow mixing red blood cells in isotonic saline with 12 volumes of deionized, endotoxin-free water and subjecting the cells to gentle agitation. It is understood that other methods of lysing the RBC's are known in the art and are within the scope of the present invention.

In order to collect the erythrocytes, the blood samples can be washed several times with an isotonic solution and the plasma can be separated by centrifugation at 3,000 rpm. Preferably, the isotonic solution used is a saline solution. Preferably, the cells are washed at least three times, rinsed between each centrifugation, and resuspended in a final volume of an equal volume of isotonic solution. The use of a sonicator may be discouraged as it makes membrane spheres (often referred to as “dust”). Agitation methods suitable for use in the present invention may include a magnetic stir bar and a mechanical rocker or shaker.

D. Separation of Stroma from Hemoglobin

The membranes of red blood cells are referred to as ghosts or stroma and contain all of the blood type antigens. Rabiner et al. first demonstrated that some of the toxic properties of hemolyzed red blood cells were related to the membrane (stroma) of red blood cells and their related lipids (Rabiner et al., J. Exp. Med. 126:1127 (1967), incorporated by reference in its entirety). The membranes can be destroyed by freezing so that storage requirements for blood may require climate controlled refrigeration. In addition, many of the human viral diseases transmitted through blood transfusions may adhere to the stroma of red blood cells. Thus, stroma-free hemoglobin (“SFH”) can be beneficial in light of the immunogenic properties, such as inflammation, agglutination, clotting, an immune mediated complement response, platelet activation, etc, of the cell membranes of red blood cells, and possibility of viral contamination.

An effective stroma-free hemoglobin blood substitute therapy can offer several advantages over conventional blood replacement therapies. Significantly, the use of stroma-free hemoglobin blood substitutes can reduce the extent and severity of undesired immune responses, and the risk of transmission of viral diseases, including hepatitis and HIV. Moreover, in contrast to the limited storage capacity of erythrocytes, stroma-free hemoglobin blood substitutes can exhibit an extended shelf life, and require less rigorous environmentally controlled storage facilities.

The stroma may be removed by ultrafiltration of the hemolysate over a 0.65 micron filter which retains the cellular components and passes the hemoglobin. Alternatively, the cellular debris may be removed by subsequent filtration through a 0.22 micron filter or a 300,00 Dalton molecular weight filter. Ultrafiltration membranes suitable for use in the present invention are commercially available from, for example, Millipore Corporation. This step can be performed at 4° C. as rapidly as possible after hemolysis of the erythrocyte. Other methods for separating Hb from the lysed RBC phase can be employed, including sedimentation, precipitation (Tye, U.S. Pat. No. 4,529,719), centrifugation or microfiltration It is understood that other methods of removing stroma are known in the art and are within the scope of the present invention.

After such treatment, the stroma-free hemolysate is concentrated by a membrane that does not allow for the passage of hemoglobin. Preferably, the stroma-free hemolysate is concentrated using a filter having a 30,000 MW cut-off. Preferably, the stroma-free hemolysate is concentrated to a 1%-25% (g/l) solution. More preferably, the stroma-free hemolysate is concentrated to about 5 to about 10%. Most preferably, the stroma-free hemolysate is concentrated to about 10%. The concentrated solution can be equilibrated with buffer and the pH is adjusted. Preferably, the pH is adjusted to a pH of 7.40. However, a pH of between about 6.5 and about 8.5 can be used in the present invention.

The concentrated Hb solution can then be directed into one or more parallel chromatographic columns to further separate the hemoglobin by high performance liquid chromatography from other contaminants such as antibodies, endotoxins, phospholipids and enzymes and viruses. Examples of suitable media include anion exchange media, cation exchange media, hydrophobic interaction media and affinity media. The chromatographic columns may contain an anion exchange medium suitable to separate Hb from non-hemoglobin proteins. Suitable anion exchange mediums include, for example, silica, alumina, titania gel, cross-linked dextran, agarose or a derivatized moiety, such as a polyacrylamide, a polyhydroxyethyl-methacrylate or a styrene divinylbenzene, that has been derivatized with a cationic chemical functionality, such as a diethylaminoethyl or quaternary aminoethyl group. A suitable anion exchange medium and corresponding eluants for the selective absorption and desorption of Hb as compared to other proteins and contaminants, which are likely to be in a lysed RBC phase, are readily determinable by one of reasonable skill in the art.

E. Removal of Phosphate Ion

Bucci et al. (U.S. Pat. No. 5,290,919) have reported that removal of organic phosphates, e.g., 2,3-diphosphoglycerate, is necessary in human hemolysates because the site of the cross-linking reaction is the same as that occupied by 2,3-diphosphoglycerate in hemoglobin. Accordingly, in some embodiments of the present invention, the stroma free human Hb. (before crosslinking) that has passed through the filter may be then treated to exchange phosphate for chloride. For this purpose, the stroma free human Hb can be passed in the absence or presence of oxygen, through an ion exchange column that has been previously prepared and equilibrated with chloride. Efficacy of this step may be measured by total inorganic phosphate analysis. Suitable ionic resins are commercially available and are within the scope of the present invention. The ionic resin removes phosphate that competes for the aspirin binding site during the reaction with BDBF.

Because inorganic phosphate interferes with the cross-linking reaction in human Hb, it may need to be removed from the human hemoglobin in order for the reaction to provide a satisfactory yield. This can be accomplished using a suitable exchange resin with chloride ion. A buffer can be provided if there is a substantial change in the exchange of the phosphate ion for the chloride ion. Preferably, phosphate buffers may not be employed during any of the processing steps of the present invention. In some embodiments of the present invention, the stroma free human Hb solution is substantially free from inorganic phosphate. Preferably, the stroma free human Hb solution of the present invention is free from inorganic phosphate. One way of removing inorganic phosphate from the stroma free human Hb solution is to pass the stroma free human Hb solution over an ion exchange matrix equilibrated with chloride. Such a process may remove phosphate by competing with phosphate for the aspirin binding site of hemoglobin. This may be done in a nitrogen atmosphere. The solution can then be concentrated to the desired range. This operation is not necessary when using bovine Hb.

Preferably, any ion removal or buffer equilibration can be performed using counter flow dialysis so as to prevent accumulation of endotoxin in the subsequent product. The material can then be sterile filtered into a suitable container. Oxygen affinity of the hemoglobin derivative of the present invention can be measured using the hemoxyanalyser (TCS-medical products) or the gill cell described by Dolman et al., Anal. Biochem. 87:127 (1978), incorporated by reference in its entirety.

F. Protecting Cysteine of the Hemoglobin

A thiol group of a cysteine moiety in a hemoglobin may bind to the nitric oxide and may result in transient or sustained changes in hydrodynamic properties of blood or vasoactive substances and may lead to hypertension. This occurrence can be avoided by protecting the thiol group of the cysteine moiety in the hemoglobin such that the resulting hemoglobin is incapable of binding with NO. The SFH of the present invention can be reacted with various reagents to result in protection of the thiol group in the cysteine moiety of the hemoglobin. The step of protecting cysteine of the hemoglobin with thiol protecting groups may be carried out before the cross linking step or after the cross linking step. In a preferred embodiment of the present invention, the step of protecting the thiol group in the cysteine moiety is carried out before the cross-linking step. In a preferred embodiment of the present invention, the step of deoxygenating the hemoglobin is carried out before the step of protecting the thiol group in the cysteine moiety. Without limiting the scope of the present invention, all the reagents known in the art for the protection of a functional group such as, but not limited to, hydroxyl, thiol, or carboxyl, are included in the present invention.

Some of the examples of the reagents include, but are not limited to, 4-pyridylmethyl chloride, alkoxyalkylchloride, dimethoxymethane, N-(hydroxymethyl)acetamide, triphenylmethyl chloride, acetyl chloride, 2-chloroacetic acid, acetic anhydride, haloacetamide such as, iodoacetamide, bromoacetamide, chloroacetamide, or fluoroacetamide, haloacetate such as iodoacetate, bromoacetate, chloroacetate, or fluoroacetate, benzyl chloride, benzoyl chloride, di-tert-butyl dicarbonate, p-hydroxyphenacyl bromide, p-actoxybenzyl chloride, p-methoxybenzyl chloride, 2,4-dinitrophenyl fluoride, tetrahydropyran, acetamidohydroxymethane, acetone, bis-carboethoxyethene, 2,2,2-trichloroethoxycarbonyl chloride, tert-butoxycarbonyl chloride, alkyl isocyanate, and alkoxyalkyl isocyanate. In a preferred embodiment, the reagent is haloacetamide. In a further preferred embodiment, the reagent is iodoacetamide. It is understood that any reagent known in the art that can be used for carboxymethylation of the thiol group in the cysteine moiety of the hemoglobin is within the scope of the present invention.

Without limiting the scope of the present invention, all the protecting groups known in the art for the protection of a functional group such as, but not limited to, hydroxyl, thiol, or carboxyl, are included in the present invention. Some of the examples of the protecting group include, but are not limited to, 4-pyridylmethyl, acetylaminomethyl, alkoxyalkyl, triphenylmethyl, carboxymethyl, acetyl, benzyl, benzoyl, tert-butoxycarbonyl, p-hydroxyphenacyl, p-acetoxybenzyl, p-methoxybenzyl, 2,4-dinitrophenyl, isobutoxymethyl, tetrahydropyranyl, acetamidomethyl, bezamidomethyl, bis-carboethoxyethyl, 2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, N-alkyl carbamate, and N-alkoxyalkyl carbamate. In a preferred embodiment, the protecting group is carboxymethyl such that the protection of the thiol group in the cysteine moiety of the hemoglobin results in a non-pyrogenic, endotoxin free, stroma free, carboxymethylated Hb (CMSFH). More generally, protection of the thiol group in cysteine(s) of hemoglobin results in a non-pyrogenic, endotoxin free, stroma free thiol blocked Hb (TBSFH).

G. Removal of Oxygen

The CMSFH can be treated under conditions sufficient to remove oxygen present in the preparation. One aspect of the present invention concerns an improved process for removing oxygen from CMSFH preparations. Without limiting the scope of the present invention, such deoxygenation can be carried out before or after any of the steps disclosed herein. For example, the deoxygenation step can be performed prior to or after the step of removing stroma, the step of removing the endotoxins, the step of thiol protection, the step of phosphate removal, the step of lysis of RBCs, or the step of cross-linking of the hemoglobin. In a preferred embodiment, such deoxygenation is performed prior to the protection of the thiol group in the cysteine moiety of the hemoglobin of the present invention. In some embodiments of the invention, the steps of the method may require more time to be completed. In such cases, deoxygenated conditions may be preferred.

The extent of deoxygenation can be measured by gas chromatograph, zirconium-based detector (e.g., a “MOCON” analyzer (Mocon, Minneapolis, Minn.), by measuring pO₂ or by measuring the spectral shift that is characteristic of deoxyhemoglobin formation. Oxygen in the hemoglobin can be removed by vacuum, or by vacuum centrifugation. The CMSFH used may be an ultrafiltrate obtained from the removal of stroma (dilute) or a retentate from the ultrafiltration of the second stage ultrafiltration conducted to concentrate the hemoglobin to approximately 10% (w/v). Either of these solutions of CMSFH obtained can be readily deoxygenated by applying a vacuum sufficient to equal the partial pressure of water at the temperature of the solution, while the solution can be centrifuged at a speed sufficient to produce a force greater than the surface tension of the solution. These are generally low speeds and can be met with preparatory centrifuges, or those of a continuous flow variety. It may be desirable to consider the geometry of the containers of the CMSFH to insure that there may be adequate surface area for gas exchange and that the temperature can be maintained and the solution not allowed to freeze.

The contactor membrane technology can be used to remove O₂ from Hb solutions. The contactor membrane technology can also be used for oxygenation where oxygen gas may be used instead of nitrogen gas. A three or four of such equipments may be attached in series for higher throughput and can be used for commercial production of deoxygenated or oxygenated hemoglobin solution. The Hb concentration affects the rate at which the dissolved O₂ is removed. As the Hb concentration is lowered the O₂ removal rate increases. The experiment may not lower O₂ concentration to <100 ppb. However, the test can be performed in the glove box to make the system gas tight. The glove box can maintain the environment at very low O₂ levels (<5 ppb). This glove box environment can provide the O₂ barrier required to ensure that no O₂ can be re-absorbed by the Hb. Hg vacuum >28.5″ (<50 mm Hg) can be used for optimum O₂ removal.

The deoxygenated, endotoxin free, stroma free, carboxymethylated Hb (dCMSFH) prepared in the manner described above may be preferably maintained in an inert environment and the pH of the preparation may be preferably adjusted to a range between 6.0 and 9, and most preferably about pH 8.8-8.9. The pH of the solution may be preferably adjusted using dilute 0.1 N citric acid or 0.1 N NaOH. Where dilution, suspension, or addition of water (including buffers, etc.) for other purposes is desired, such water may be deoxygenated and be free of endotoxin. All subsequent steps may be carried out in the absence of oxygen, maintained by what ever means is desired. As indicated above, a preferred method involves the use of nitrogen positive pressure environmental glove box, however, other inert gases (e.g., argon) may be equivalently employed in lieu of nitrogen.

H. Cross-Linking with BDBF and Reaction with PLP

Stroma-free Hb can be prevented to dissociate into αβ dimers by conjugating Hb to another molecule such as polyethylene glycol (PEG), which may form a hydrophilic shield around the Hb molecule and may simultaneously increase its size which in turn may increase its circulatory half-life. Hb can also be cross-linked intramolecularly to prevent dissociation of the tetramer into αβ dimers and/or crosslinked intermolecularly to form polymers which may also increase the oxygen carrier's size and thus increase its circulatory half-life. Using site-specific cross-linking reagents, it may be possible to form intramolecular covalent bonds, which may convert Hb into a stable tetramer, thus preventing its dissociation into αβ dimers. Alternatively, the use of non-specific cross-linkers such as glutaraldehyde may lead to non-specific covalent bonding between amino acid residues residing within and between Hb tetramers. This may lead to the formation of hemoglobin polymers (polyHb) of various molecular weights.

Chemical reagents with multi-aldehyde functionalities which can be used as cross-linking agents include but are not limited to, molecules such as glutaraldehyde, ring-opened raffinose and dextran. In the case of aldehydes, the formation of covalent cross-links may be initiated by the carbonyl group of the aldehyde reacting with an amino group present in the Hb tetramer. Polymerization of Hb into larger molecules may increase the half-life of the polyHb with respect to tetrameric Hb and prevents Hb dissociation into αβ dimers. PolyHb may eventually get filtered out of the systemic circulation through the kidneys, the lymphatic system, and the reticuloendothelial system (RES).

Examples of suitable cross-linking agents include polyfunctional agents that will cross-link Hb proteins, such as glutaraldehyde, succindialdehyde, activated forms of polyoxyethylene and dextran, α-hydroxy aldehydes, such as glycolaldehyde, N-maleimido-6-aminocaproyl-(2′-nitro,4′-sulfonic acid)-phenyl ester, m-maleimidobenzoic acid-N-hydroxysuccinimide ester, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N-succinimidyl(4-iodoacetyl)aminobenzoate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl 4-(p-maleimidophenyl)butyrate, sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N′-phenylene dimaleimide, and compounds belonging to the bis-imidate class, the acyl diazide class or the aryl dihalide class, among others. The saccharides can be used as cross-linking agents. The examples of saccharides include, but are not limited to, monosaccharides (galactose, glucose, methylglucopyranoside, and mannitol), disaccharides (lactose, maltose, cellobiose, sucrose, and trehalose), a trisaccharide (raffinose) and polysaccharides (dextrans with molecular weights of 15,000 and 71,000 Da).

A suitable amount of a cross-linking agent may be that amount which may permit intramolecular cross-linking to stabilize the Hb and also intermolecular cross-linking to form polymers of Hb, to thereby increase intravascular retention. Without limiting the scope of the present invention, various strategies can be employed to cross-link Hb with desirable molecular weight distributions and oxygen binding properties. The type and concentration of both crosslinking and quenching agents, the duration of the cross-linking/polymerization reaction, and utilization of reducing agents are all possible variables that can be modified in these reactions to engineer the molecular weight distribution, oxygen binding properties, and metHb levels of cross-linked Hb dispersions.

High performance liquid chromatography (HPLC) Size Exclusion Chromatography (SEC) can be used to determine the percent of total cross-linked Hb, percent of cross-linked tetramer, or percent of crosslinked higher order species of Hb/polyHb dispersions. A salt such as, MgCl₂ can serve to dissociate any non-cross-linked tetrameric Hb into αβ dimers, while cross-linked tetrameric Hb may remain intact. Hence, non-cross-linked Hb may elute in a separate peak away from intramolecularly cross-linked Hb.

The cross-linking of hemoglobin may be conducted in the absence of oxygen. Inorganic phosphate, which binds tightly to the hemoglobin molecule and interferes with the cross-linking reaction, may be removed to increase yield. Endotoxins, which bind tightly to the hemoglobin molecule and become a hepatic toxin when the hemoglobin is metabolized, may not be allowed to contact the hemoglobin.

The dCMSFH as disclosed herein is cross-linked to form cross-linked tetrameric hemoglobin. In some embodiments of the present invention, the dCMSFH is cross-linked with bis 3′,5′ dibromo salicyl fumarate (BDBF) (Tye, U.S. Pat. No. 4,529,719, hereby incorporated by reference in its entirety). To accomplish this, BDBF cross-linker may be added with stirring to provide mixing to the dCMSFH preparation at a molar ratio of BDBF cross-linker:dCMSFH of greater than 1:1. Preferably, the molar ratio of BDBF cross-linker:dCMSFH is 2:1. Prior to such addition, the pH of the dCMSFH preparation may be adjusted to match that of the BDBF. The pH of the reaction mixture is carefully maintained by the addition of acid or base since the solution is not buffered. The reaction is permitted to go to completion.

Pyridoxal-5-phosphate (PLP) has the ability to modify many hemoglobins. Although the properties of dXCMSFH from human hemoglobin benefit from the pyridoxal-5-phosphate reaction, dXCMSFH from bovine hemoglobin does not require this step. PLP modifies human hemoglobin by introducing a negative charge near a penultimate β chain histidine residue and by removing a positive charge at the amino terminal end of the same chain. These charge changes stabilize a new molecular configuration that is similar to the hemoglobin-DPG (diphosphoglycerate) complex. Significantly, the hemoglobin of this new configuration has an oxygen affinity resembling that of native hemoglobin within the red cell. The product may have one or two PLP molecules attached per tetramer. In prior PLP-hemoglobin preparations the intravascular retention time was too short to permit such preparations to be acceptable as a resuscitation fluid. Additionally, they were found to cause osmotic diuresis.

Accordingly, after the cross-linking reaction has been completed, where using human Hb, pyridoxal-5-phosphate (PLP) is added to the deoxygenated, endotoxin free, stroma free, carboxymethylated cross-linked human Hb (dXCMSFH) preparation. The PLP is reduced with sodium borohydride and then permitted to react with the dXCMSFH and to form dXCMSFH-pyridoxal-5′-phosphate (dXCMSFH-PLP) using the methods described by Benesch et al. (Benesch et al., Biochemistry 11:3576 (1972); Benesch et al., Biochem. Biophys. Res. Commun. 63(4): 1123-9 (1975); Benesch et al., Methods Enzymol. 76:147-59 (1981); Benesch et al., J. Biol. Chem. 257(3):13204 (1982); Schnackerz et al.; and, J. Biol. Chem. 258(2):872-5 (1983), all of which references are incorporated herein by reference in their entirety) with the change that all reagents are free of endotoxin and oxygen and the reaction occurs in the absence of oxygen. This treatment is not necessary when using bovine Hb.

I. Equilibration

The dXCMSFH can be equilibrated with Ringer's lactate or Ringer's acetate solution. After equilibration, the solution can be sterile filtered into suitable infusion containers. Infusion containers suitable for use in the present invention may include, but are not limited to, sterile IV bags. Preferred infusion containers may prevent gas exchange (i.e., impermeable to oxygen) and the dXCMSFH can be stored in the absence of oxygen. This is expected to prevent the heme oxidation to form methemoglobin.

Analysis

The dXCMSFH as disclosed herein can be analyzed at any step of the process of making it. Hemoglobin can be analyzed, for example, but not limited to, after removing stroma, after removing endotoxin, after lysis, after removing oxygen, after protection of thiol group in the cysteine moiety, or after cross-linking etc. The hemoglobin can be analyzed for purity, absorbance, structure, p50, nitric oxide binding capacity, white blood cell (WBC) count, microorganism growth in the hemoglobin solution, cross-linking, amino acid analysis, protein analysis, or effect of refrigeration or storage. Various analytical techniques are known in the art and are all within the scope of the present invention. Some of the examples of the analytical techniques are provided herein but they are not limiting to the scope of the present invention.

Mass Spectrometry (MS)

There are many types of mass spectrometers and sample introduction techniques which allow a wide range of analyses and they are all included herein. In some preferred embodiments of the present invention, the technique used is mass spectrometry. Mass spectrometers may consist of three distinct regions: Ionizer, Ion Analyzer, and Detector. Ionization methods include, but are not limited to, electron impact (EI), chemical ionization (CI), electrospray (ESI), fast atom bombardment (FAB), and matrix assisted laser desorption (MALDI). Analyzers include but are not limited to, quadrupole, sector (magnetic and/or electrostatic), time-of-flight (TOF), and ion cyclotron resonance (ICR). The mass spectrometer may be coupled with LC or GC.

Liquid chromatography mass spectrometry (LC/MS or LC-MS) separates compounds chromatographically before they are introduced to the ion source and mass spectrometer. It differs from GC/MS in that the mobile phase is liquid, usually a combination of water and/or organic solvents, instead of gas. Typically, an electrospray ionization source is used in LC/MS.

Ion mobility spectrometry/mass spectrometry (IMS/MS) is a technique where ions are first separated by drift time through some pressure of neutral gas given an electrical potential gradient before being introduced into a mass spectrometer. The drift time is a measure of the radius relative to the charge of the ion. The duty cycle of IMS (time over which the experiment takes place) is longer than most mass spectrometers such that the mass spectrometer can sample along the course of the IMS separation. This produces data about the IMS separation and the mass-to-charge ratio of the ions in a manner similar to LC/MS. However, the duty cycle of IMS is short relative to liquid chromatography or gas chromatography separations and can thus be coupled to such techniques producing triply hyphenated techniques such as LC/IMS/MS.

Tandem mass spectrometry (MS/MS) involves multiple steps of mass selection or analysis, usually separated by some form of fragmentation. A tandem mass spectrometer is one capable of multiple rounds of mass spectrometry. For example, one mass analyzer can isolate one compound from many entering a mass spectrometer. A second mass analyzer then stabilizes the compound ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID). A third mass analyzer then catalogs the fragments produced from the compound. Tandem MS can also be done in a single mass analyzer over time as in a quadrupole ion trap. There are various methods for fragmenting molecules for tandem MS, including collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD).

In certain embodiments, the methods utilize an ESI-MS detection device. An ESI-MS combines the ESI system with mass spectrometry and may further utilize a time-of-flight (TOF) mass spectrometry system. In TOF-MS, ions are generated by whatever ionization method is being employed, such as ESI, and a voltage potential is applied. The potential extracts the ions from their source and accelerates them towards a detector. By measuring the time it takes the ions to travel a fixed distance, the mass to charge ratio of the ions can be calculated. TOF-MS can be set up to have an orthogonal-acceleration (OA). In addition to the MS systems disclosed above, other forms of ESI-MS include quadrupole mass spectrometry, ion trap mass spectrometry, orbitrap mass spectrometry, Fourier transform ion cyclotron resonance (FTICR-MS), and hybrid combinations of these mass analyzers.

In Quadrupole mass spectrometry, applied voltages affect the trajectory of the ions traveling down the flight path. Only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other ions are thrown out of their original path. A mass spectrum is obtained by monitoring the ions passing through the quadrupole filter as the voltages on the rods are varied. Ion trap mass spectrometry uses rf fields to trap ions. A quadrupole ion trap uses three electrodes in a small volume. The mass analyzer consists of a ring electrode separating two hemispherical electrodes. A linear ion trap uses end electrodes to trap ions in a linear quadrupole. A mass spectrum is obtained by changing the electrode voltages to eject the ions from the trap.

Orbitrap mass spectrometry uses spatially defined electrodes with DC fields to trap ions. Ions are constrained by the DC field and undergo harmonic oscillation. The mass is determined based on the axial frequency of the ion in the trap. FTICR mass spectrometry is a mass spectrometric technique that is based upon an ion's motion in a magnetic field. Once an ion is formed, it eventually finds itself in the cell of the instrument, which is situated in a homogenous region of a large magnet. The ions are constrained in the XY plane by the magnetic field and undergo a circular orbit. The mass of the ion can be determined based on the cyclotron frequency of the ion in the cell. Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS) includes linear TOF-MS and reflectron TOF-MS. Both linear and reflectron MALDI-TOF-MS can be utilized for molecular weight determinations of molecular ions.

MALDI TOF-MS is a tool in the biosciences for obtaining both accurate mass determinations and primary sequence information. The MALDI technique has increased the upper mass limit for mass spectrometric analyses of biomolecules to over 300,000 Da and has enabled the analyses of large biomolecules by mass spectrometry to become easier and more sensitive. Improved mass resolution in MALDI TOF-MS has been obtained by the utilization of a single-stage or a dual-stage reflectron (RETOF-MS). In the reflectron mass spectrum, the isotopic multiplet is well resolved producing a full width half maximum (FWHM) mass resolution of about 3400. Mass resolutions up to 6000 (FWHM) have been obtained for peptides up to about 3000 Da with RETOF-MS. Enhancing the mass resolution can also increase the mass accuracy when determining the ion's mass.

Both linear and reflectron MALDI-TOF-MS can be utilized for molecular weight determinations of molecular ions and enzymatic digests leading to structural information of proteins. These digests are typically mass analyzed with or without purification prior to molecular weight determinations. Varieties of methodologies have been developed to obtain primary sequence information for proteins and peptides utilizing MALDI TOF-MS. Two different approaches can be taken. The first method is known as protein ladder sequencing and can be employed to produce structurally informative fragments of the analyte prior to insertion into the TOF mass spectrometer and subsequent analysis. The second approach utilizes the phenomenon of metastable ion decay that occurs inside the TOF mass spectrometer to produce sequence information.

The ladder sequencing with TOF-MS consists of either a time-dependent or concentration-dependent chemical degradation from either the N- or C-terminus of the protein/peptide into fragments, each of which differs by one amino acid residue. The mixture is mass analyzed in a single MALDI-TOF-MS experiment with mass differences between adjacent mass spectral peaks corresponding to a specific amino acid residue. The order of occurrence in the mass spectrum defines the sequence of amino acids in the original protein/peptide.

Post-source decay with RETOF-MS MALDI is an ionization technique that produces intact protonated pseudomolecular ion species. A significant degree of metastable ion decay occurs after ion acceleration and prior to detection. The ion fragments produced from the metastable ion decay of peptides and proteins typically include both neutral molecule losses (such as water, ammonia and portions of the amino acid side chains) and random cleavage at peptide bonds.

In-source decay with linear TOF-MS is an alternative approach to RETOF-MS for studying metastable ion decay of MALDI generated ions. Primary structural information for peptides and proteins can be obtained by this method. Coherent mass spectral peaks are produced from these metastable decayed ions giving rise to significant structural information for peptides and proteins.

Another proteomic technology involved in quantitative analysis of protein mixtures is known as surface-enhanced laser desorption ionization—time of flight (SELDI-TOF). This technique utilizes stainless steel or aluminum-based supports, or chips, engineered with chemical (hydrophilic, hydrophobic, pre-activated, normal-phase, immobilized metal affinity, and cationic or anionic) or biological (antibody, antigen binding fragments (e.g. scFv), DNA, enzyme, or receptor) bait surfaces of 1-2 mm in diameter. These varied chemical and biochemical surfaces allow differential capture of proteins based on the intrinsic properties of the proteins themselves. Solubilized tissue or body fluids in volumes as small as 0.1 μl are directly applied to these surfaces, where proteins with affinities to the bait surface may bind. Following a series of washes to remove non-specifically or weakly bound proteins, the bound proteins are laser desorbed and ionized for MS analysis. Masses of proteins ranging from small peptides of less than 1000 Da up to proteins of greater than 300 kDa are calculated based on time-of-flight. As mixtures of proteins may be analyzed within different samples, a unique sample fingerprint or signature may result for each sample tested. Consequently, patterns of masses rather than actual protein identifications can be produced by SELDI analysis. These mass spectral patterns can be used to differentiate patient samples from one another, such as diseased from normal.

UV-Vis

In some embodiments of the present invention, optical absorption spectroscopy (UV/VIS) has been used to determine the absorbance range for the hemoglobin. UV/VIS plays a role for the determination of concentrations of macromolecules such as proteins. Organic dyes can be used to enhance the absorption and to shift it into the visible range (e.g. coomassie blue reagents). Understanding the forces that govern the interaction of proteins with one another assists in the understanding of such processes as macromolecular assembly, chaperone-assisted protein folding and protein translocation. Resonance Raman spectroscopy (RRS) is a tool which can be used to study molecular structure and dynamics. Resonance Raman scattering requires excitation within an electronic absorption band and results in a large increase of scattering. This approach may help to investigate specific parts of macromolecules by using different excitation wavelengths.

Liquid Chromatography (LC)

Liquid chromatography is a tool for isolating proteins, peptides, and other molecules from complex mixtures. In some embodiments of the present invention, LC has been used for separation, purification and analysis of the hemoglobin. Examples of LC can be affinity chromatography, gel filtration chromatography, anion exchange chromatography, cation exchange chromatography, diode array-LC and high performance liquid chromatography (HPLC).

Gel filtration chromatography separates proteins, and peptides on the basis of size. Molecules may move through a bed of porous beads, diffusing into the beads to greater or lesser degrees. Smaller molecules may diffuse further into the pores of the beads and therefore move through the bed more slowly, while larger molecules may enter less or not at all and thus move through the bed more quickly. Both molecular weight and three dimensional shapes contribute to the degree of retention. Gel Filtration Chromatography may be used for analysis of molecular size, for separations of components in a mixture, or for salt removal or buffer exchange from a preparation of macromolecules.

Affinity chromatography is the process of bioselective adsorption and subsequent recovery of a compound from an immobilized ligand. This process allows for the specific and efficient purification of many diverse proteins and other compounds.

Ion exchange chromatography separates molecules based on differences between the overall charges of the proteins. It is usually used for protein purification but may be used for purification of peptides, or other charged molecules. The protein of interest may have a charge opposite that of the functional group attached to the resin in order to bind. Because this interaction can be ionic, binding can take place under low ionic conditions. Elution can be achieved by increasing the ionic strength to break up the ionic interaction, or by changing the pH of the protein.

HPLC can be used in the separation, purification and detection of hemoglobin of the present invention. Use of reversed-phased chromatography (RPC) can be utilized in the process of protein structure determination. The normal procedure of this process can be 1) fragmentation by proteolysis or chemical cleavage; 2) purification; and 3) sequencing. A common mobile phase for RPC of peptides can be, for example, a gradient of 0.1% trifluoroacetic acid (TFA) in water to 0.1% TFA in an organic solvent, such as acetonitrile, since the organic solvent 1) solubilizes the protein or peptide, 2) allows detection at approximately 230-240 nm, and 3) can evaporate away from the sample.

The use of size-exclusion chromatography (SEC) and ion-exchange chromatography (IEC) can be used in determining the structure of the hemoglobin of the present invention. Full recovery of activity after exposure to the chromatography may be achieved, and SEC columns can be diverse enough to allow fractionation from 10 to 1000 kilodaltons. The use of gradient elution with the IEC column may be favorable because of equivalent resolution as polyacrylamide gel electrophoresis (PAGE) and increased loading capability when compared to SEC. In liquid affinity chromatography (LAC) interaction may be based on binding of the protein due to mimicry of substrate, receptor, etc. The protein may be eluted by introducing a competitive binding agent or altering the protein configuration which may facilitate dissociation. HPLC may be coupled with MS.

Diode array detector-liquid chromatography (DAD-LC) provides complete, multiple spectra for each HPLC peak, which, by comparison, can provide indication of peak purity. These data can also assign presence of tyr, trp, phe, and possibly others (his, met, cys) and can quantitate these amino acids by 2nd derivative or multi-component analysis. By a post-column derivatization, DAD-LC can also identify and quantitate cys, his and arg in individual peptides. Thus, it is possible to analyze for 6 of the 20 amino acids of each separated peptide in a single LC run, and information can be obtained about presence or absence of these amino acids in a given peptide in a single step. This is assisted by knowing the number of residues in each peptide.

Electrophoresis

Electrophoresis can be used to for the analysis of the hemoglobin of the present invention. Electrophoresis can be gel electrophoresis or capillary electrophoresis.

Gel Electrophoresis: Gel electrophoresis is a technique that can be used for the separation of proteins. Separation of large (macro) molecules may depend upon two forces: charge and mass. When a biological sample, such as proteins, is mixed in a buffer solution and applied to a gel, these two forces may act together. The electrical current from one electrode may repel the molecules while the other electrode may simultaneously attract the molecules. The frictional force of the gel material acts as a “molecular sieve,” separating the molecules by size. During electrophoresis, macromolecules are forced to move through the pores when the electrical current is applied. Their rate of migration through the electric field depends on the strength of the field, size and shape of the molecules, relative hydrophobicity of the samples, and on the ionic strength and temperature of the buffer in which the molecules are moving. After staining, the separated macromolecules in each lane can be seen in a series of bands spread from one end of the gel to the other. Using this technology it is possible to separate and identify protein molecules that differ by as little as a single amino acid. Also, gel electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight. Electrofocusing or isoelectric focusing is a technique for separating different molecules by their electric charge differences (if they have any charge). It is a type of zone electrophoresis that takes advantage of the fact that a molecule's charge changes as the pH of its surroundings changes.

Capillary Electrophoresis: Capillary electrophoresis is a collection of a range of separation techniques which may involve the application of high voltages across buffer filled capillaries to achieve separations. The variations include separation based on size and charge differences between analytes (termed capillary zone electrophoresis (CZE) or free solution CE (FSCE)), separation of neutral compounds using surfactant micelles (micellar electrokinetic capillary chromatography (MECC) or sometimes referred to as MEKC) sieving of solutes through a gel network (capillary gel electrophoresis, GCE), separation of cations (or anions) based on electrophoretic mobility (capillary isotachophoresis, CITP), and separation of zwitterionic solutes within a pH gradient (capillary isoelectric focusing, CIEF). Capillary electrochromatography (CEC) can be an associated electrokinetic separation technique which involves applying voltages across capillaries filled with silica gel stationary phases. Separation selectivity in CEC can be a combination of both electrophoretic and chromatographic processes. Many of the CE separation techniques rely on the presence of an electrically induced flow of solution (electroosmotic flow, EOF) within the capillary to pump solutes towards the detector. GCE and CIEF are of importance for the separation of biomolecules such as proteins.

Operation of a CE system may involve application of a high voltage (typically 10-30 kV) across a narrow bore (25-100 μm) capillary. The capillary may be filled with electrolyte solution which conducts current through the inside of the capillary. The ends of the capillary can be dipped into reservoirs filled with the electrolyte. Electrodes made of an inert material such as platinum can also be inserted into the electrolyte reservoirs to complete the electrical circuit. A small volume of sample can be injected into one end of the capillary. The capillary may pass through a detector, usually a UV absorbance detector, at the opposite end of the capillary. Application of a voltage may cause movement of sample ions towards their appropriate electrode usually passing through the detector. A plot of detector response with time can be generated which is termed an electropherogram. A flow of electrolyte, known as electroendosmotic flow, EOF, may result in a flow of the solution along the capillary usually towards the detector.

Nuclear Magnetic Resonance (NMR)

NMR can be used for the analysis of the hemoglobin of the present invention. NMR spectroscopy is capable of determining the structures of hemoglobin at atomic resolution. In addition, it is possible to study time dependent phenomena with NMR, such as intramolecular dynamics in macromolecules, reaction kinetics, molecular recognition or protein folding. Heteronuclei like ¹⁵N, ¹³C and ²H, can be incorporated in proteins by uniformly or selective isotopic labeling. Spectra from these samples can be drastically simplified. Additionally, some new information about structure and dynamics of macromolecules can be determined with these methods.

X-Ray Crystallography

X-ray crystallography can be used for the analysis of the hemoglobin of the present invention. X-ray crystallography is a technique in which the pattern produced by the diffraction of X-rays through the closely spaced lattice of atoms in a crystal is recorded and then analyzed to reveal the nature of that lattice. This generally leads to an understanding of the material and molecular structure of a substance. The spacings in the crystal lattice can be determined by using Bragg's law. The electrons that surround the atoms, rather than the atomic nuclei themselves, are the entities which physically interact with the incoming X-ray photons. This technique can be used to determine the structure of the hemoglobin of the present invention. X-ray diffraction is commonly carried out using single crystals of a material, but if these are not available, microcrystalline powdered samples may also be used which may require different equipment.

Arrays

Arrays can be used for the analysis of the hemoglobin of the present invention. Arrays involve performing parallel analysis of multiple samples against known protein targets. The development of various microarray platforms can enable and accelerate the determination of protein abundance, localization, and interactions in a cell or tissue. Microarrays provide a platform that allows identification of protein interaction or function against a characterized set of proteins, antibodies, or peptides. Protein-based chips array proteins on a small surface and can directly measure the levels of proteins in tissues using fluorescence-based imaging. Proteins can be arrayed on either flat solid phases or in capillary systems (microfluidic arrays), and several different proteins can be applied to these arrays. Nonspecific protein stains can be then used to detect bound proteins.

Amino Acid Analysis

In some embodiments, amino acid analysis (AAA) is a technique used in the analysis of the hemoglobin of the present invention. AAA is a process to determine the quantities of each individual amino acid in a protein. There can be four steps in amino acid analysis: hydrolysis, derivatization, separation of derivatized amino acids, and data interpretation and calculations.

In the hydrolysis step, a known amount of internal standard (norleucine) may be added to the sample. Since norleucine does not naturally occur in proteins, is stable to acid hydrolysis and can be chromatographically separated from other protein amino acids, it makes an efficient internal standard. The molar amount of internal standard can be approximately equal to that of most of the amino acids in the sample. The sample, containing at least 5 nmoles of each amino acid (i.e. 10 μg of protein) can be then transferred to a hydrolysis tube and dried under vacuum. The tube can be placed in a vial containing HCl and a small amount of phenol and the protein is hydrolyzed by the HCl vapors under vacuum. The hydrolysis is carried out for about 24 hours at about 110° C. Following hydrolysis, the sample can be then dissolved in distilled water containing EDTA (to chelate metal ions) and approximately 1 nmole of each amino acid can be placed on a glass amino acid analyzer sample slide. Hydrolysis can have varying effects on different amino acids.

In the derivatization step, the free amino acids cannot be detected by HPLC unless they have been derivatized. Derivatization can be performed automatically on the amino acid analyzer by reacting the free amino acids, under basic conditions, for example, with phenylisothiocyanate (PITC) to produce phenylthiocarbamyl (PTC) amino acid derivatives. This process may take approximately 30 minutes per sample. A standard solution containing a known amount (500 pmol) of 17 common free amino acids can also be loaded on a separate amino acid analyzer sample spot and derivatized. This can be used to generate a calibration file that can be used to determine amino acid content of the sample. Following derivatization, a methanol solution containing the PTC-amino acids can be transferred to a narrow bore HPLC system for separation.

The PTC-amino acids can be separated on a reverse phase C18 silica column and the PTC chromophore can be detected at 254 nm. All of the amino acids may elute in approximately 25 minutes. The buffer system used for separation can be for example, 50 mM sodium acetate at pH 5.45 as buffer A and 70% acetonitrile/32 mM sodium phosphate at pH 6.1 as buffer B. The program can be run using a gradient of buffer A and buffer B.

Chromatographic peak areas can be identified and quantitated using a data analysis system that can be attached to the amino acid analyzer system. A calibration file can be used that can be prepared from the average values of the retention times (in minutes) and areas in (Au) of the amino acids in 10 standard runs. Since a known amount of each amino acid is loaded onto the analyzer, a response factor ((Au/pmol) can be calculated. This response factor can be used to calculate the amount of amino acid (in pmols) in the sample.

Alternatively, the classical method of amino acid analysis of Moore and Stein using ninhydrin may be used.

Formulations

The dXCMSFH of the present invention may be incorporated in conventional pharmaceutical formulations (e.g. injectable solutions) for use in treating mammals in need thereof. Pharmaceutical compositions can be administered by subcutaneous, intravenous, or intramuscular injection, or as large volume parenteral solutions and the like. In some embodiments, dXCMSFH of the present invention is formulated by encapsulating Hb within liposomes. Liposome encapsulation is often used in drug delivery to reduce the toxicity of encapsulated therapeutic agents, as well as to increase drug half-life. The liposome-encapsulated hemoglobin (LEHb) encases Hb in a structure physiologically similar to RBCs, thus preventing Hb dissociation and its rapid clearance in the blood stream. The half-life of LEHb dispersions is dependent on the surface chemistry of the bilayer, as well as the bilayer surface charge and the vesicle size distribution. Hence, decreasing vesicle size and modifying the vesicle surface can significantly increase the circulatory lifetime. Surface conjugation of liposomes with polyethylene-glycol (PEG) can extend the half-life. The uptake of liposomes by the reticuloendothelial system (RES) affects the LEHb concentration that can be safely administered, since overloading the RES would impair the immune system.

The dXCMSFH of the present invention can also be formulated into other blood substitute formulations. Such formulations can include other components in addition to the dXCMSFH. For example, a parenteral therapeutic composition can comprise a sterile isotonic saline solution. The formulations can be either in a form suitable for direct administration, or in a concentrated form requiring dilution prior to administration. The formulations of the present invention can thus contain between 0.001% and 90% (w/v) dXCMSFH. In some embodiments of the present invention, the extracelluar hemoglobin solution of dXCMSFH of the present invention may contain from about 5 percent to about 20 percent, from about 5 percent to about 17 percent, from about 8 to about 14 percent, and about 10 percent hemoglobin in solution (% weight per volume). The selection of percent hemoglobin depends on the oncotic properties of the chosen hemoglobin product. The hemoglobin solutions formulated for use in the present invention may be normo-oncontic to hyperoncotic. The percent hemoglobin may be adjusted to obtain the desired oncotic pressure for each indication.

dXCMSFH of the present invention can be used in compositions useful as substitutes for red blood cells in any mammal that uses red blood cells for oxygen transport. The mammals include but are not limited to, human, livestock such as cattle, cat, horse, dog, sheep, goat, pig etc. Preferably, the mammal is human.

A dose of the dXCMSFH of the present invention can be from about 1 to about 15,000 milligrams of hemoglobin per kilogram of patient body weight. When used as an oxygen carrying composition, or as a blood substitute, the dosage may range between 100 to 7500 mg/kg patient body weight, 500 to 5000 mg/kg body weight, or 700 to 3000 mg/kg body weight. Thus, a dose for a human patient might be from a gram to over 1000 grams. It will be appreciated that the unit content of active ingredients contained in an individual dose of each dosage form need not in itself constitute an effective amount, as the necessary effective amount could be reached by administration of a number of individual doses. The selection of dosage depends upon the dosage form utilized, the condition being treated, and the particular purpose to be achieved according to the determination of those skilled in the art.

For use in the present invention, dXCMSFH of the present invention can be dialyzed or exchanged by ultrafiltration into a physiologically acceptable solution. The dXCMSFH of the present invention may be formulated at a concentration of 50-150 g/l. The solution may comprise a physiologically compatible electrolyte vehicle isosmotic with whole blood and which may maintain the reversible oxygen-carrying and delivery properties of the hemoglobin. The physiologically acceptable solution can be, for example, physiological saline, a saline-glucose mixture, Ringer's acetate, Ringer's solution, lactated Ringer's solution, Locke-Ringer's solution, Krebs-Ringer's solution, Hartmann's balanced saline, heparinized sodium citrate-citric acid-dextrose solution, and polymeric plasma substitutes, such as polyethylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol and ethylene oxide-propylene glycol condensates.

Each formulation according to the present invention may additionally comprise inert constituents including pharmaceutically-acceptable carriers, diluents, fillers, salts, and other materials well-known in the art, the selection of which depends on the dosage form utilized, the condition being treated, the particular purpose to be achieved according to the determination of the ordinarily skilled artisan in the field and the properties of such additives. For example, the hemoglobin solution of the present invention in addition to dXCMSFH may include 0-200 mM of one or more physiological buffers, 0-200 mM of one or more carbohydrates, 0-200 mM of one or more alcohols or poly alcohols, 0-200 mM of one or more physiologically acceptable salts, and 0-1% of one or more surfactants, 0-20 mM of a reducing agent. The hemoglobin solution of the present invention in addition to dXCMSFH may include, 0-50 mM sodium gluconate, 0-50 mM of one or more carbohydrates (e.g. glucose, mannitol, sorbitol or others known to the art), 0-300 mM of one or more chloride salts and, optionally, 0-0.5% surfactant, e.g. Tween. TM. [polysorbate 80], and/or 0-20 mM N-acetyl cysteine.

Administration of the dXCMSFH of the present invention can occur for a period of seconds to hours depending on the purpose of the hemoglobin usage. For example, when used as an oxygen carrier for the treatment of severe hemorrhage, the usual time course of administration is as rapidly as possible. Typical infusion rates for hemoglobin solutions as oxygen therapeutics can be from about 100 ml/hour to about 3000 ml/hour, from about 1 ml/kg/hour to about 300 m/kg/hour, or from about 1 ml/kg/hour to about 25 ml/kg/hour.

Suitable compositions can also include 0-200 mM of one or more buffers (for example, acetate, phosphate, citrate, bicarbonate, or Goode's buffer). Salts such as sodium chloride, potassium chloride, sodium acetate, calcium chloride, magnesium chloride can also be included in the compositions of the invention. The salt can be in concentrations of 0-2 M.

In addition, the compositions of the invention can include one or more carbohydrate (for example, reducing carbohydrates such as glucose, maltose, lactose or non-reducing carbohydrates such as sucrose, trehalose, raffinose, mannitol, isosucrose or stachyose) and one or more alcohol or poly alcohol (such as polyethylene glycols, propylene glycols, dextrans, or polyols). The concentration of carbohydrate or alcohol can be 0-2 M.

The dXCMSFH of the present invention can also contain one or more surfactant and 0-200 mM of one or more chelating agent (for example, ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), o-phenanthroline, diethylamine triamine pentaacetic acid (DTPA also known as pentaacetic acid) and the like). The surfactant can be 0.005-1% of the composition. The compositions of the invention can be at pH of about 6.5-9.5. In some embodiments, the composition may contain 0-150 mM NaCl, 0-10 mM sodium phosphate, 0.01-0.1% surfactant, and/or 0-50 μM of one or more chelating agents at pH 6.6-7.8. The formulation may contain 5 mM sodium phosphate, 150 mM NaCl, 0.025% to 0.08% polysorbate 80, and/or 25 μM EDTA at pH 6.8-7.6.

Additional additives to the formulation can include anti-bacterial agent, oncotic pressure agent (e.g. albumin or polyethylene glycols) and other formulation acceptable salt, sugar and other excipients known in the art. Each formulation according to the present invention can additionally comprise constituents including carriers, diluents, fillers, salts, and other materials well-known in the art, the selection of which depends upon the particular purpose to be achieved and the properties of such additives which can be readily determined by one skilled in the art. The compositions of the present invention can be formulated by any method known in the art. Such formulation methods include, for example, simple mixing, sequential addition, emulsification, diafiltration and the like.

Packaging and Storage of dXCMSFH, dCMSFH and dTBSFH

The dXCMSFH, dCMSFH, and dTBSFH of the present invention may be stored in conventional, and preferably oxygen impermeable containers (for example, stainless steel tanks, oxygen impermeable plastic bags, or plastic bags overwrapped with low oxygen permeably plastic bags wherein an oxygen scavenger is placed between the internal plastic bag and the overwrapped plastic bag). In some preferred embodiments, the dXCMSFH, dCMSFH, or dTBSFH of the present invention is stored in the absence of oxygen. The dXCMSFH, dCMSFH, or dTB SFH may be oxygenated prior to use such as, by way of example only, oxygenating before using in the catheter for cardiac therapy. In some embodiments, the dXCMSFH, dCMSFH, or dTBSFH can be stored in oxygen permeable or oxygen impermeable (“anoxic”) containers in an oxygen controlled environment. Such oxygen controlled environments can include, for example, glove boxes, glove bags, incubators and the like. Preferably the oxygen content of the oxygen controlled environment is low relative to atmospheric oxygen concentrations (see, Kandler, R. L. et al., U.S. Pat. No. 5,352,773; herein incorporated by reference). In some embodiments of the present invention, the dXCMSFH, dCMSFH, or dTBSFH can be packaged in sealed Tyvek or Mylar (polyethylene terephthalate) bags or pouches. In some embodiments, the dXCMSFH, dCMSFH, or dTBSFH of the present invention can be lyophilized and stored as a powder. The preparations may be stored at room or elevated temperature (Kandler et al., PCT Publication No. WO 92/02239; Nho, PCT Publication No. WO 92/08478, both herein incorporated by reference), or more preferably under refrigeration. In some embodiments, the dCMSFH or dTBSFH may be stored in HyClone BioProcess Containers™ for ease of shipping and further handling.

Where the package is an oxygen impermeable film, the container can be manufactured from a variety of materials, including polymer films, (e.g., an essentially oxygen-impermeable polyester, ethylene vinyl alcohol (EVOH), or nylon), and laminates thereof. Where the container is an oxygen impermeable overwrap, the container can be manufactured from a variety of materials, including polymer films, (e.g., an essentially oxygen-impermeable polyester, ethylene vinyl alcohol (EVOH), or nylon) and laminates, such as a transparent laminate (e.g. a silicon oxide or EVOH containing-laminate) or a metal foil laminate (e.g., a silver or aluminum foil laminate). The polymer can be a variety of polymeric materials including, for example, a polyester layer (e.g., a 48 gauge polyester), nylon or a polyolefin layer, such as polyethylene, ethylene vinyl acetate, or polypropylene or copolymers thereof.

The containers can be of a variety of constructions, including vials, cylinders, boxes, etc. In a preferred embodiment, the container is in the form of a bag. A suitable bag can be formed by continuously bonding one or more (e.g., two) sheets at the perimeter(s) thereof to form a tightly closed, oxygen impermeable, construction having a fillable center. In the case of laminates comprising polyolefins, such as linear low density, low density, medium or high density polyethylene or polypropylene and copolymers thereof, the perimeter of the bag may be bonded or sealed using heat. It is well within the skill of the art to determine the shape of the bag and the appropriate temperature to generate a tightly closed, oxygen and/or moisture impermeable construction. Where the container is a film, such as a polyester film, the film can be rendered essentially oxygen-impermeable by a variety of suitable methods. The film can be laminated or otherwise treated to reduce or eliminate the oxygen permeability.

In some embodiments, one or more antioxidants, such as ascorbate (Wiesehahn, G. P. et al., U.S. Pat. No. 4,727,027; and, Kerwin, B. D. et al., U.S. Pat. No. 5,929,031); gluathione, acetylcsyteine, methionine, tocopherol, butyl hydroxy toluene, butyl hydroxy anisole, or pholic compounds. (Osterber et al., PCT Publication No. WO 94/26286; and, Kerwin, B. D. et al., U.S. Pat. No. 5,929,031) may be added to further stabilize the dXCMSFH, dCMSFH, and dTBSFH (all such references herein incorporated by reference). Alternatively, and more preferably, the dXCMSFH of the present invention can be lyophilized and stored as a powder, or can be packaged in sealed Tyvek, or Mylar (polyethylene terephthalate) bags or pouches. Packaging such as, Kerwin, B. D. et al., U.S. Pat. No. 5,929,031, is herein incorporated by reference. In some embodiments, the dXCMSFH, dCMSFH, and dTBSFH in such storage containers may be subjected to irradiation or other sterilization treatment sufficient to extend the shelf-life of the compositions. An oxygen scavenger such as n-acetyl-cycleine may be included in the formulation.

The dXCMSFH, dCMSFH, and TBSFH of the present invention may be stored at suitable storage temperatures for periods of two years or more, and preferably for periods of two years or more, when stored in a low oxygen environment. Suitable storage temperatures for storage of one year or more are between about 0° C. and about 40° C. The preferred storage temperature range is between about 0° C. and about 25° C. The process of making dXCMSFH, dCMSFH, and dTBSFH of the present invention includes maintaining the steps of the process under conditions sufficient to minimize microbial growth, or bioburden, such as maintaining temperature at less than about 20° C. and above 0° C.

Methods of Use

The dXCMSFH of the present invention may be used to form pharmaceutical compositions that may be administered to recipients, for example, by infusion, by intravenous or intra-arterial injection, or by other means. The dXCMSFH formulations of the present invention can be used in compositions useful as substitutes for red blood cells in any application where red blood cells are used. Such compositions of the present invention formulated as red blood cell substitutes can be used for the treatment of hemorrhage where blood volume is lost and both fluid volume and oxygen carrying capacity must be replaced. Moreover, because the dXCMSFH of the present invention can be made pharmaceutically acceptable, the formulations of the present invention can be used not only as blood substitutes that deliver oxygen but also as simple volume expanders that provide oncotic pressure due to the presence of the large hemoglobin protein molecule. The dXCMSFH of the present invention can thus be used as replacement for blood that is removed during surgical procedures where the patient's blood is removed and saved for reinfusion at the end of surgery or during recovery (e.g., acute normovolemic hemodilution or hemoaugmentation, etc.).

A typical dose of the dXCMSFH of the present invention as a blood substitute is from 10 mg to 7 grams or more of extracellular hemoglobin per kilogram of patient body weight. Thus, a typical dose for a human patient might be from a few grams to over 350 grams. It will be appreciated that the unit content of active ingredients contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount could be reached by administration of a plurality of administrations as injections, etc. The selection of dosage depends upon the dosage form utilized, the condition being treated, and the particular purpose to be achieved according to the determination of the ordinarily skilled artisan in the field.

In some embodiments of the invention, a solution of dXCMSFH will contain about 5% to about 25% dXCMSFH by weight for administration to a mammal. In some preferred embodiments of the invention the solution of dXCMSFH will contain about 7% to about 15% dXCMSFH by weight for administration to a mammal. In some other preferred embodiments of the invention, a solution of dXCMSFH will be 10% by weight of dXCMSFH for administration to a mammal. In some embodiments of the invention, a dose to be administered to a mammal contains about 7 g of dXCMSFH. In some embodiments of the invention a dose to be administered to a mammal contains about 1 g of dXCMSFH. In some embodiments of the invention, an exemplary unit of production for use in a therapeutic setting is a container with 500 ml of a 0.5 mmol solution of dXCMSFH (about 64 g/L, or about 6.4% by weight in solution). The larger unit solutions may be used for replacement of blood for a number of therapeutic interventions. The smaller unit solutions may be used for labelling and diagnostic purposes, as well as therapeutic interventions. The smaller unit solutions may, in a preferred embodiment, contain a solution of dXCMSFH of up to 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 15%, 16%, 17%, 18%, 19%, 20%, 21, 22%, 23%, 24%, or up to 25% by weight of dXCMSFH.

Administration of the dXCMSFH of the present invention can occur for a period of seconds to hours depending on the purpose of the hemoglobin usage. For example, as a blood delivery vehicle, the usual time course of administration is as rapid as possible. Typical infusion rates for hemoglobin solutions as blood replacements can be from about 100 ml to 3000 ml/hour. However, when used to stimulate hematopoiesis, administration can last only seconds to five minutes and therefore administration rates can be slower because the dosage of the dXCMSFH of the present invention may be much less than dosages that can be required to treat hemorrhage.

In some embodiments, the dXCMSFH of the present invention can be used to treat anemia as caused by renal failure, diabetes, AIDS, chemotherapy, radiation therapy, hepatitis, G.I. blood loss, iron deficiency, menorrhagia, and the like, by providing additional oxygen carrying capacity in a mammal that is suffering from anemia, by stimulating hematopoiesis, and by serving as an adjuvant to erythropoietin therapy. Likewise, the dXCMSFH of the present invention can be used to provide additional oxygen carrying capacity to a mammal (such as an athlete, soldier, mountaineer, aviator, smoke victim, etc.) desiring such additional oxygen carrying capacity. The formulations of the present invention thus are useful in treating carbon monoxide poisoning and its concurrent hypoxia and ischemia. The dXCMSFH of the present invention can be used for applications requiring administration to a mammal of high volumes of hemoglobin as well as in situations where only a small volume of the hemoglobin of the present invention is administered. The dXCMSFH of the invention can be used in applications during surgery where large volumes of blood are normally lost, or in treatment of trauma victims who have lost large volumes of blood. This can include both civilian accidents and military situations. The dXCMSFH of the present invention may be used as a blood substitute in veterinary clinical applications. Thus, the term “mammal” refers to animals and humans.

In addition, because the distribution in the vasculature of the dXCMSFH is not limited by viscosity or by the size of red blood cells, the compositions of the present invention can be used to deliver oxygen to areas that red blood cells cannot penetrate. These areas can include any tissue areas that are located downstream of obstructions to red blood cell flow, such as areas downstream of thrombi, sickle cell occlusions, arterial occlusions, angioplasty balloons, surgical instrumentation, tissues that are suffering from oxygen starvation or are hypoxic, and the like. Additionally, all types of tissue ischemia can be treated using the methods of the instant invention. Such tissue ischemias include, for example, stroke, emerging stroke, transient ischemic attacks, myocardial stunning and hibernation, acute or unstable angina, emerging angina, infarct, and the like. The recovery of tissues from physical damage such as burns can also be accelerated by pretreatment with the hemoglobin of the present invention, which allows increased perfusion and oxygenation of the tissues.

The dXCMSFH of the present invention can be used for the treatment of sickle cell anemia patients. Sickle cell anemia patients in vasoocclusive crisis are currently treated by transfusion of red blood cells in conjunction with dilution and pain management. The dXCMSFH of the present invention may not only deliver oxygen thereby preventing further sickling (as do red blood cells), they may also penetrate vessels already occluded with deformed red cells to better alleviate pain and minimize tissue damage. Also, frequent transfusions in the sickle cell anemia population may result in alloimmunization to red cells and to platelets, an adverse effect that would be avoided by use of hemoglobin of the present invention. The dXCMSFH of the present invention offer a significant therapeutic advantage in treatment of sickle cell anemia patients, since they elicit a lesser degree of vasoconstriction or none at all. This is an advantage in the treatment of vasoocclusive crisis, and is also an advantage in other treatments of sickle cell anemia patients in situations where there is a risk of sudden onset of vasoocclusive crisis. For example, dXCMSFH of the present invention may be used in place of packed red cells for preoperative transfusion of sickle cell anemia patients to minimize risk of anesthesia. The dXCMSFH of the present invention may also be administered periodically to minimize risk of stroke.

The dXCMSFH of the present invention contains iron, and as such, may be detected via MRI (magnetic resonance imaging). Thus, in some embodiments, the present invention contemplates the use of dXCMSFH as an imaging agent. Although humans have four main red cell antigens (A, B, O and Rh), accounting for 12 main blood types, non-human animals exhibit far greater blood type diversity. The existence of larger numbers of blood types has complicated the use of donated blood in non-human animal transfusions (Hale, A. S., Vet Clin North Am Small Anim Pract 25:1323-1332 (1995); and, Harrell, K. A., et al., Vet Clin North Am Small Anim Pract 25:1333-1364 (1995), both references herein incorporated by reference. The dXCMSFH formulations of the present invention, which can be used regardless of the blood type of the recipient, thus finds additional utility as a blood substitute for non-human animals (e.g., dogs, horses, cats, etc.).

The present invention also concerns implantable delivery devices (such as cartridges, implants, etc.) that contain dXCMSFH, and that are capable of releasing dXCMSFH into the circulation in response to a sensed need for increased oxygen carrying capacity. In some embodiments, such devices can deliver dXCMSFH at a constant rate, so as to facilitate erythropoiesis (either alone, or in combination with erythropoietin). In some embodiments, the devices can be controlled by sensing means (such as electronic probes of hemoglobin, O₂ level, CO₂ level, etc.) so as to deliver dXCMSFH at a rate commensurate with the patient's oxygen carrying capacity needs. Such sensing means may themselves be implantable, or part of the implanted device, or may be located extracorporeally. In some embodiments, such devices may be used to accomplish or facilitate the hemo-diagnosis of individuals.

The dXCMSFH of the present invention may also be used to form non-pharmaceutical compositions that can be used, for example, as reference standards for analytical instrumentation needing such reference standards, reagent solutions, control of gas content of cell cultures; for example by in vitro delivery of oxygen to a cell culture, and removal of oxygen from solutions. Additionally, the dXCMSFH of the present invention may be used to oxygenate donated tissues and organs during transport.

The dXCMSFH can be used in treating mammals, such as humans, who have HIV/SARS, hepatitis, cancer, cardiovascular diseases, orthopedics or trauma, where such mammal is in need of oxygen carrying capacity.

The dXCMSFH of the present invention may be used to scavenge endotoxin from surfaces or liquids. The invention thus contemplates devices, such as cartridges, filters, beads, columns, tubing, and the like that contain the dXCMSFH of the present invention. Liquids, such as water, saline, culture medium, albumin solutions, etc., may be treated by passage over or through such devices in order to remove endotoxin that may be present in such liquids, or to lessen the concentration of endotoxin present in such liquids. The dXCMSFH of such devices is preferably immobilized (as by affinity, ionic, or covalent bonding, etc.) to solid supports present in such devices. In some embodiments, the dXCMSFH is bound to beads that may be added to the liquids being treated, and then subsequently removed (as by filtration, or affinity immobilization). In some embodiments, the beads may be of ferromagnetic or paramagnetic metal, or may be themselves magnetic, such that they may be readily separated from the treated liquid by magnetic means.

The CMSFH, TBSFH, and XCMSFH of the present invention may be adsorbed or bound to toweling, air filters, etc. so that endotoxin present on surfaces or in air may be removed or its concentration lessened. In a similar manner, the dCMSFH, dTBSFH, and dXCMSFH of the present invention can be used to remove oxygen from solutions requiring the removal of oxygen, and as reference standards for analytical assays and instrumentation. The CMSFH, TBSFH, and XCMSFH of the present invention can also be used in vitro to enhance cell growth in cell culture by maintaining oxygen levels.

The re-oxygenated XCMSFH of the present invention can be used for the use in visualizing intravascular space. Present optical techniques for the observation of vascular walls are relegated to non-optical techniques due to the opaque effects of the transfusion of red blood cells. The XCMSFH of the present invention may not only deliver oxygen thereby preventing ischemia, but they may also present an optically translucent field of view to allow for visualization of tissue beds in situ for the determination of pathology; cancer, vulnerable plaques, lipid damage, stent placement, etc, using visible light in the red wavelength band, for example, to illuminate the targeted feature.

While the XCMSFH because of its size can perfuse, it can be a carrier of oxygen to allow for the placement of oxygen in tissue beds that would normally be dependent upon diffusion due to the poor perfusion of bulky large red blood cells, such is the case in poorly vascularized tumor tissue beds of cancer cells where appropriate use of the invention can allow for an increase in oxygen tension and allow for more effective use of radiation therapy and for the enhancement of oxygen dependent pharmaceutical agents.

EXAMPLE 1

Method

Bovine Blood Collection: The bovine blood collection is performed on site at a local meat processing facility. The subject is a purebred black angus steer that is raised by a local cattle rancher. The subject is placed in a cattle shoot located in the slaughter room of the meat processing facility. The subject is then executed by the “mushroom stunner” technique. Subject is raised head first to a level of approximately 1 meter above ground to expose the carotid arteries. The arteries are then severed and blood is transfused into a 1 gallon collection container which may hold 100 mL of 6% sodium ethylenediaminetetraacetic acid (EDTA) solution. The collection containers are placed in a cooler, packed in ice, and transported to the laboratory. Total collection time in the meat processing facility is about 15 minutes. Transport time back to the laboratory takes about 25 minutes.

Cleaning Whole Blood: Once the whole bovine arterial blood arrives in the laboratory it is divided into Batch A and Batch B. Batch A consists of 2200 mL of whole blood and is washed in the haemonetics Cell Saver 5 to obtain concentrated red blood cells free of platelets, clotting factors, extra cellular potassium, anticoagulants, and cell stroma (Method A). Batch B consists of 1800 mL of whole blood and is washed with a Millipore 0.65 u filter (Method B). The batches are then refrigerated for 8 hours. After 8 hours both batches are passed through a Baxter leuko-reducing filter, which also removes viral materials. Batch A yields 1500 mL of RBCs while Batch B yields 1200 mL of RBCs. Samples are extracted from each batch throughout the course of cleaning.

Comparison of Method A and Method B: The Method A and Method B purifications are run side by side to compare efficiency in releasing Hb while removing stroma and preventing leucocyte lysis. The data, vida infra, show equivalency between methods. Either method will provide purified materials of acceptable quality, and a combination of both methods may also be envisioned to be used in the methods of the invention. Lysing and Filtering: The 1500 mL of RBCs from Batch A are diluted with 6000 mL of DI water. After allowing the cells 45 seconds to lyse, 750 mL of 9% N saline (NS) is added to the solution to minimize the lysing of any leukocytes present. The 1200 mL of RBCs from Batch B are lysed with 4800 mL of DI water. No saline is added to Batch B at this point. Next, both batches are passed through a 0.22 micron pellicon filter. Once the hemoglobin has been filtered out and collected in a separate flask, it is passed through a second 10K Dalton Pellicon filter. This filters out any saline present and discards it as waste into a separate beaker. The pure hemoglobin get recirculated into the original flask and is concentrated to the desired percentage, such as 13.5%. Additional samples are taken after the lysing step.

Blocking Thiols with Iodoacetamide (IAM): After the samples are concentrated to 13.5% Hb, oxygen is removed as described above, and the pH is adjusted to 7.4 with 0.1M sodium phosphate buffer. Oxygen remaining is <10 ppb. Two molar equivalents of IAM per mole of dSFH and the reaction is allowed to proceed for one hour. Progress of the reaction is monitored by an iodide electrode. The product, dCMSFH is a stable intermediate after removal of iodide ion and unreacted IAM by ultrafiltration using PBS. The stable intermediate dCMSFH is packaged in oxygen barrier containers and can be safely stored at room temperature.

Deoxygenation and Cross-Linking: The stable intermediate product, dCMSFH, is again placed in an oxygen free (<10 ppm) environment and dissolved oxygen removed to a level of less than 0.010 ppm. Membrane contactor technology can be the method utilized to deoxygenate the hemoglobin in a controlled atmosphere with an oxygen level of less than 10 ppm. An initial oxygen saturation reading is taken for both batches using a polorgraphic dissolved oxygen probe. Batch A has an initial reading at 4 mg/L. Batch B has an initial reading at 7 mg/L. The hemoglobin is pumped and recirculated through the membrane contactor using a peristaltic pump at a flow rate of 600 mL/min with applied vacuum pressure of >28.5 in/Hg. The final oxygen saturation level for both batches is <0.01 ppm. The batches are then pH adjusted to 8.4 with 0.5M NaOH for cross-linking. Once the pH is adjusted, 2.94 g of bis-3,5-dibromosalicyl fumarate (DBSF) is added to Batch A while 1.47 g is added to Batch B. Once cross-linking is complete, pH is adjusted with 0.5M citric acid back to 7.4 and the batches are stored in air-tight containers in the refrigerator.

To determine the percentage of hemoglobin that cross-links, an electrophoresis is performed. The samples are prepared in the normal way. The samples are then placed in the Beckman CoulterPA-800 Proteomics instrument for analysis where results reveal a 95% success rate. The samples are also in a Hemox Analyzer for the recording of blood oxygen equilibrium curves based on dual wavelength spectrophotometry.

Analytical Results:

A. Cell Saver 5 removal of plasma proteins: Cell Saver 5 from Haemonetics is used to purify erythrocytes from freshly collected anticoagulated bovine blood. It may be desirable not to fracture either leucocytes or erythrocytes since, it is used for erythrocyte salvage in the operating room to purify blood from the operating theatre and recycle for infusion.

After passing through a coarse filter of 150 μm, the cells are washed in a spinning bowl holding 225 ml of packed red blood cells and washed with 3 liters of saline in a reverse flow from the outside edge of the bowl towards the center. The centrifugation is gentle, and some of the WBCs are eluted in the wash, which may be desirable. Table 1 shows the progress of serum protein removal at 500 ml increments. It is a continuous flow technique. Sample in Table 1 is the sample volume applied to the bowl. Progress is followed by reading the protein concentration at 280 mμin a spectrophotometer. The data points in Table 1 are taken at the indicated points during the washing process. The results indicate that when a full bowl of red cells is washed with 3 liters of NS, there is a greater than 3 log reduction in serum proteins. This also infers a greater than 3 log reduction in viruses, prions, etc that are not bound to red cell membranes. All values in Table 1 are corrected for dilution.

TABLE 1 Cell Saver 5 removal of plasma proteins A₂₈₀ of plasma 277.35 A₂₈₀ of filtrate after 500 cc NS 43.20 A₂₈₀ of filtrate after 1000 cc NS 7.69 A₂₈₀ of filtrate after 1500 cc NS 0.86 A₂₈₀ of filtrate after 2000 cc NS 0.05

B. Millipore 0.65 μm filter removal of plasma proteins: The Millipore filter is used to purify erythrocytes from serum proteins. Freshly collected anticoagulated blood as indicated in A (paragraph 00156) is passed through a 150 μm filter. The material is then filtered with a tangential flow membrane that will pass plasma proteins but retain cellular components such as, leucocytes and erythrocytes. The tangential flow membrane can be slower but it may require less labor as it can run unattended. It can be more suitable for scaleup. Other types of large scale centrifuges may be used. The results of this continuous diafiltration are shown in Table 2, where all results are corrected for dilution. This method reveals a greater than 3 log reduction in plasma proteins, which also implies a similar log reduction in viruses, prions, etc.

TABLE 2 A₂₈₀ of plasma in 1000 cc blood 221.6 A₂₈₀ of filtrate after 1000 NS 82.1 A₂₈₀ of filtrate after 2000 NS 26.5 A₂₈₀ of filtrate after 3000 NS 8.07 A₂₈₀ of filtrate after 4000 NS 2.76 A₂₈₀ of filtrate after 5000 NS 0.81 A₂₈₀ of filtrate after 6000 NS 0.29 A₂₈₀ of filtrate after 7000 NS 0.102

C. Evaluation of leucocyte loss/removal: It is desirable during the preparation of hemoglobin to remove any WBC's so that the proteolytic enzymes of the granulocytes are not present in the hemoglobin solution. Thus at the stage of removing plasma proteins it is desirable to remove the WBC's without causing their lysis. The Cell Saver 5 technology can lose some of the WBCs in the floating buffy coat during centrifugation. However, the tangential flow membrane may retain all of the WBC's so a loss may mean that cell lysis of the WBC's had occurred. Table 3 shows evaluation of the conservation of WBC's after filtration with Cell Saver and Millipore. When appropriately corrected for volume, both methods provide adequate protection of leucocyte lysis.

TABLE 3 Evaluation of leucocyte loss/removal Initial Final WBC % Sample WBC (Vol Adj) Recovery Cell Saver 5 5.79 × 10³ 6.10 × 10³ 100% Millipore 0.65 5.79 × 10³ 5.63 × 10³ 100%

D. Leucoreduction using a Pall filter: White blood cells can cause febrile reactions into human recipients when present in transfused packed RBCs. It is desirable to use a leucoreduction filter which can pass the rbcs but markedly reduce the number of WBCs. A larger prototype than that used for single human unit of packed cells is used to evaluate the leucoreduction. The leukoreduction of chilled washed bovine erythrocytes can be achieved in about 12 hours. The results are shown in Table 4. A 3 log reduction in WBC's is achieved by the passage of the red cell suspension through a leucocyte reduction filter. This is an alternative to the method wherein RBCs are selectively lyzed in the presence of WBC without lyzing the WBC, which are subsequently removed by filtration. This selective lyzing is discussed more fully below (see Example 2).

TABLE 4 Leucocyte Reduction Filter Initial Vol Adjusted % Log₁₀ Sample WBC Final WBC Removal Removal 12 hr Cold Bovine 6.13 × 10³ 0.028 99.6% 3

E. Erythrocyte/Leucocyte differential hypotonic lysis: Plasma free blood cells are equilibrated with NS, and then diluted into 4 volumes of DI. This can result in the fracturing of the plasma free blood cells by the hypotonic lysis. The cells can explode as they are fractured by the rapid uptake of water. Red blood cells can be lysed in about 30 seconds, while WBC's can take longer. The lysis of the WBC's can be prevented by restoring the isotonic state of the WBC with hypertonic saline addition to make the equivalent concentration of NS. The following experiment is conducted to see if the red blood cells can be lysed without the fracturing of the WBC's. The hemoglobin can then be removed by a 0.22 μm filter as filtrate while the retentate would concentrate the stroma, red cell membranes and the unlysed WBC's. FIG. 2 depicts the time course of a lysis experiment showing resistance of WBC lysis for up to 5 minutes. Erythrocyte lysis can be stopped during the two minute period before appreciable leucocyte lysis occurs.

F. Stroma separation from hemoglobin after hypotonic lysis: The contents of the erythrocyte are about 98.5% in pure hemoglobin, with some small amount of other proteins including carbonic anhydrase. The lysis of washed plasma free erythrocytes can be stopped by the restoration of normal saline tonicity. The filtration of the lysed plasma free erythrocytes allows the passage of the hemoglobin and retention of stroma and leucocytes. This can yield pure hemoglobin. Microscopic analysis of 10 ml spun samples does not reveal any cellular debris. Phosphatidylcholine is a component of the cell wall. Preliminary test for phosphotidylcholine is conducted using chromatographic techniques. Phoshatidylcholine is found to be below the limit of detection.

G. Concentration of hemoglobin to 14% solution: The hemoglobin is filtered on a 0.22 μm filter. The hemoglobin concentration is between 2-3%. The hemoglobin solution is concentrated on a tangential flow membrane with a MW cutoff of 30 kD. The filtrate is free from color, while the retentate hemoglobin is concentrated to the desired range. Preferably, the range is about 14%.

H. Determination of cross link using SDS gel electrophoresis: SDS can denature proteins to form long rods covered by negative charges of the carboxyl group at neutral pH. Proteins can then be separated by size exclusion using electrophoresis since the manifold excess of negative charge by the SDS can dwarf the charge heterogeneity of the native proteins. The Beckman Coulter PA-800 provides rapid record of the gel electrophoresis by using the absorption at 216 nm for the peptide bond. Standard protein mixtures can be used to calibrate the column in use, as shown in FIG. 3. Of particular interest are the sizing standards for the region between 10 KDa and 100 KDa.

Native hemoglobin run on SDS gel electrophoresis can give two peaks with almost baseline separation between them. The first peak has been shown to be the alpha chain and the second the beta chain of hemoglobin. They can occur in almost equal amounts as there are equal number of chains in the molecule. This is illustrated in FIG. 4A, which is an expanded region of the size separation overlays shown in FIG. 4B. Native hemoglobin appears as the green trace (rt13.98 and rt14.14), crosslinked hemoglobin according to the present invention (dXCMSFH) appears as the red trace, and size standards are blue. The overlaid spectra shown in FIG. 4A are slightly offset for ease of viewing. The expanded region illustrated is clustered about the 20 KDa size standard, as would be expected for the two alpha and beta subunits of native hemoglobin. The experiment shown here for the cross-linked material is taken from a midpoint in the process, when most of the alpha chains have already crosslinked, but significant amounts of beta chain still remain unreacted.

In FIG. 4B, the overlay of the full width chromatographs are shown, again with native hemoglobin in green, the crosslinking experiment in red, and the size standards in blue. The fumaryl crosslinking of the two alpha chains yields a pair of peptides tethered together and thus will appear at later elution times than the unreacted beta chains. The series of three new major product peaks at a higher molecular weight of about 36,000-45,000 KDA are seen in FIG. 4B atrt of 16 to 17 min. The product peaks may not be quantified as the size standards are all single peptide chains and cannot be extended for quantification for the dimerized products of this reaction. However, it can be seen that three higher molecular weight products are being formed.

There is a formation of beta to beta crosslinks in addition to the alpha alpha crosslinks. There is a small amount of material at 90,000 KDa which can indicate the formation of a small number of inter molecular crosslinks.

I. Optimization of crosslink using pH and excess crosslink agent: Table 5 shows a grid of increasing pH vs. mole ratio of crosslink agent (DBSF) for a two hour period. The results show the % of alpha chain unreacted at different pH and molar DBSF. The pH is maintained constant with the titration of the acid produced by reaction with NaOH. After the two hour period the production of acid has long since ceased. Results of crosslink are determined using the PA-800, looking at residual uncrosslinked alpha chains.

TABLE 5 pH vs. Molar Ratio DBSF pH Molar DBSF 7 7.5 8 8.2 8.4 1 21% 1.2 19% 1.5  3% 2.0 38% 11% 2.5% 1.7% 1.5%  2.5 1.7% 

J. Reaction with iodoacetamide (IAM): The iodoacetamide reaction is followed with an iodide specific electrode, since one of the byproducts is iodide ion. Excess IAM is removed by equilibration with Ringer's Acetate and diafiltration. A two molar excess can be used. Table 6 shows a grid of time course of the iodoacetamide reaction for bovine Hb vs. the moles of IAM reagent used in the IAM reaction. The results show the amount of free sulfhydryl per mole of Hb.

TABLE 6 Reaction of Bovine Hb with IAM Moles Time IAM 0 15 30 45 60 75 90 120 2 2 1.0 0.5 0.5 4 0.5 0.2 0.1 <0.1

K. Modified hemoglobin affinity for oxygen: Hemoglobin has an ability to bind and release oxygen under physiological conditions as a function of the partial pressure of oxygen in the system. Hemox-Analyzer (made by TCS Corporation) allows the determination of the hemoglobin oxygen dissociation curve. Other methods to obtain hemoglobin oxygen dissociation curve may not be reproducible, accurate and easy to perform. The hemoglobin oxygen dissociation curves can be altered by changing in pH, temperature, CO₂ concentration, species of hemoglobin, variant of human hemoglobin, hemolyzed hemoglobin, and the like. The shape of the curve and the shift of the curve along the X axis can describe the ability of the hemoglobin to load and unload oxygen. The information can be useful in research for blood and modified hemoglobin as it is an in vitro test of in vivo function. It can measure the ability of hemoglobin to load and unload oxygen. A short hand description of the entire hemoglobin dissociation curve can be given by the p50 for O₂, the partial pressure of oxygen in mm of Hg, which can cause the hemoglobin to be half saturated with oxygen.

Chemical modifications to hemoglobin or genetic variants to hemoglobin can cause the p50 to decrease, i.e. bind oxygen more tightly at any given oxygen pressure. In vivo this can mean less oxygen to perfused tissue.

The Hemox-Analyzer relies upon the change in color of blood (hemoglobin) that is arterial (oxygenated, red) and venous (less oxygenated, blue), and an oxygen electrode. A small dilute sample is prepared in a special spectrophotometric cell that has a small orifice in the bottom that allows a purified gas to be slowly bubbled through a stirred solution and also fitted with an oxygen electrode. The entire cell is precisely temperature controlled at 37° C., to equilibrate to body temperature. The outputs of the spectrophotometer and the oxygen electrode are fed into a computer, which then analyzes the numerous data points and plots the curves. At the beginning of a plot the sample is fully oxygenated by bubbling pure oxygen through the sample until the oxygen saturation is greater than the fraction of oxygen in air (21%), then the gas bubbler is switched to nitrogen and the removal of oxygen begins. Oxygen equilibrium curves are thus generated for a variety of blood and Hb samples.

The p50 for human hemoglobin in RBCs can be about 28. The p50 falls to about 14 when RBCs are separated from the 2,3DPG which forms a salt bridge in the red cell to decrease the oxygen affinity. The p50 for bovine hemoglobin whether in the red cell or in free solution is about 25 to about 40 depending on the pH and the concentration of CO₂. FIG. 5 shows oxygen affinity curves for bovine whole blood, stroma free Hb, cross linked dXCMSFH of the invention, and fresh human blood. In the crosslinked hemoglobin, the crosslinking locks the hemoglobin in the tense state and therefore loses the sigmoidal curve. At lower pO₂, when O₂ is delivered to the tissues, the crosslinked hemoglobin delivers more oxygen as compared to bovine whole blood, stroma free Hb, and fresh human blood. The p50 of crosslinked hemoglobin is higher than that of human hemoglobin, and bovine hemoglobin. The p50 value for bovine whole blood is 24.73, for stroma free Hb is 21.20, for fresh human blood is 23.72 and for dXCMSFH is 32.43.

Results. The deoxygenated, endotoxin free, stroma free, carboxymethylated cross-linked Hb (dXCMSFH) of the present invention is a stabilized tetramer of bovine hemoglobin that is locked in the tense state, so called T state. The dXCMSFH can have a p50 similar to hemoglobin within normal human red blood cells or, as shown in FIG. 5, higher. Thus, an equal amount of hemoglobin from a human red blood cell and hemoglobin from dXCMSFH, can carry the same amount of oxygen leaving the lung. However, dXCMSFH can deliver slightly more oxygen before its venous return, based upon the information obtained from the Hemox-Analyzer.

L. Assay for Endotoxin with Results. The final product, ready for infusion, must be endotoxin free. Endotoxin is actually material from bacterial cell walls, and is responsible for initiation of a fever in the recipient, in low doses; while higher levels will initiate a more serious constellation of symptoms. The LAL (Limulus Ameobocyte Lysate) kinetic-turbidimetric assay was chosen over other assays, such as the chromogenic and the gel-clot, because of its reproducible results and high degree of sensitivity.

The potency of Control Standard Endotoxin (CSE) used for routine testing is determined by comparison with Reference Standard Endotoxin (RSE), EC5, Lot F manufactured by the USPC. It is necessary to perform this comparison whenever an endotoxin other than the Referenced endotoxin is to be used for creating spikes and curves in routine testing. This is a consequence of the fact that different lots of CSE have markedly different potencies. The RSE/CSE comparison is performed by comparing one vial of RSE to four vials of the same lot of CSE and calculating an average potency. A standard curve is assayed in triplicate, with a coefficient of correlation of −0.98 or less required for qualification. Numerous CSE standard curves are run and one standard curve is archived for future testing. Inhibition/enhancement studies are performed on all products to be tested with the LAL assay.

I. Reagent and Equipment Preparation

All reagents used for the kinetic-turbidimetric assay are used according to the specific manufacturers' instructions with the following two exceptions: 1) the LAL is reconstituted with 5 ml of Pyrosol™ reconstitution buffer instead of LAL Reagent Water and 2) the CSE is not reconstituted with exactly 5 ml of LAL Reagent Water. The reconstitution of LAL with buffer is performed to overcome extreme enhancement of the LAL assay by pure hemoglobin solutions. The CSE is reconstituted with an amount of water which will yield a final solution concentration of 1000 EU/ml. The amount to be added is determined by standardizing the CSE against the USPC RSE and may be more or less than the 5 ml recommended by Associates of Cape Cod. This yields a constant CSE solution concentration and prevents recalculation of endotoxin spikes every time the CSE lot changes. All other reagents are used as directed.

All glassware is depyrogenated by heating to 180° C. for four hours. All dilutions and solution transfers are performed under a class 100 laminar flow hood. All pipette tips used are sterile and pyrogen-free.

II. Determination of CSE Potency

One vial of RSE (10,000 EU/vial by definition) was reconstituted with 5 ml of LAL Reagent Water to yield a 2,000 EU/ml solution. Two RSE curves were run to cover full range of current Q.C. testing. The mid-range curve contained the following concentrations (EU/ml): 1.0, 0.5, 0.25, 0.125, 0.0625, and 0.03125. The low range curve consisted of the following concentrations (EU/ml): 0.04, 0.02, 0.01, 0005, 0.00025, and 0.001. These curves were run in duplicate, linear regression was performed to determine the slope and Y-intercept of the curves, and the curves were archived for the purpose of comparison with the CSE curves.

After the RSE curves had been run, 4 vials of CSE (500 ng/vial) were reconstituted with an amount of water which will yield a final solution concentration of 1000 EU/each. Dilutions of each vial were prepared in each of the two ranges so that at least three concentrations of the CSE curve would fall directly on the RSE curve. The mid-range curve contained the following concentrations (ng/ml): 0.1, 0.05, 0.025, 0.0125, 0.00625 and 0.003125. The low-range curve consisted of the following concentrations (ng/ml): 0.004, 0.002, 0.001, 0.0005, 0.00025, and 0.0001. These curves were run in duplicate and the onset times were interpolated off the corresponding RSE curve.

The endotoxin concentration in EU/ml for each CSE standard was divided by the corresponding concentration in ng/ml. Any onset times which did not fall directly on the RSE curve, indicated on the raw data by an asterisk, were not included in the calculations. The resulting EU/ng potencies for each standard were averaged to determine the CSE potency in each range. The two range potencies were then averaged to determine the potency of the CSE through the entire range of 1.0 to 0.001 EU/ml. The overall CSE potency was then used to calculate the amount of LAL Reagent Water to be added to each CSE Vial in the lot to yield a 1000 EU/ml solution according to the following calculations:

${\frac{500\mspace{11mu}\frac{ng}{vial}}{1000\mspace{11mu}\frac{E\; U}{ml}} \times {potency}\mspace{11mu}\frac{EU}{ng}} = {{water}\mspace{14mu}\frac{ml}{vial}}$ III. Test Method

100 μl of LAL is added to a depyrogenated 10×75 mm culture tube containing 400 μl of sample. The tube is vortexed gently for approximately 2 seconds and placed in the incubation module of the LAL device. Each tube is added individually in this manner. Timing is initiated for each tube as the bottom of the tube actuates a mechanical switch. Tubes are incubated at 37.0±0.5° C. throughout the test. No readings are taken for the first 60 seconds; this allows time for the contents of the tube to come to temperature and for air bubbles to disperse. For 60 to 120 seconds after tube insertion, photodetectors in each well take 7 readings 10 seconds apart. The readings are then averaged and taken to represent 100% transmittance. This zeroing period eliminates data errors due to tube imperfections and sample color or endogenous turbidity. Subsequent readings are taken every ten seconds, converted to transmittance, and then to optical density (OD). From 400 to 550 seconds, the OD values collected are averaged and then subtracted from all subsequent OD values for the test. (This baseline correction compensates for the OD of the background.)

Onset time, T_(o), is defined as the number of seconds between placement of a sample in and incubating well and the development of an optical density of 20 mAU. The endotoxin level is determined by comparing the onset times to an archived standard curve of log₁₀(T_(o)) versus log₁₀ (known endotoxin concentration).

A small-programmed-computer is used to collect data from the LAL device. The LAL device software stores the OD values in data files and performs data analysis upon command such as onset time correction, linear regression on standards, and endotoxin concentration determination.

All in-process samples are tested in duplicate, unspiked and spiked, with a four-lambda spike, where lambda equals the lowest standard on the standard curve. In addition, a four-lambda spike in LAL Reagent Water and unspiked LAL Reagent Water are tested. All final product samples are tested unspiked and spiked, in triplicate.

Results. The levels of endotoxin in dXCMSFH are below 1 EU per/ml. In preferred embodiments of the process, the elimination of endotoxins to a level below 0.01 EU are achieved and allow for complex usage and for larger volumes. This results in readings of 0.1 EU per ml to below 0.02 EU per ml.

M. Determination of Residual Hemoglobin that remains without a crosslink. HPLC on a Biorad Bio-Sil® SEC-12.5-5 column of the reaction mixture using 0.5M NaCl solution as a buffer under conditions that would otherwise not denature the hemoglobin secondary structure, is a way to examine the amount of unreacted material, since under these conditions the equilibrium would favor the alpha beta dimer with a molecular weight of 32 KDa, which would be expected at greater retention times than seen for any peak in this experiment. As seen in FIG. 6, the HPLC trace shows the major peak at 64 KDa, with a minor peak at 128 KDa. There is no material at later elution times, and hence no materials with lower molecular weight. This experiment demonstrates the complete absence of unreacted hemoglobin and illustrates that most of the material is the stabilized tetramer of Hb with a molecular weight of 64 KDa.

O. Phospholipid Level Reduction. Another key element to the dXCMSFH of the invention is the low level of phospholipids present. Phospholipids derive from the surface lipid layer of the red cells which acted as source for the hemoglobin. The steps of processing given above remove these unwanted lipids, thus eliminating the problems associated with their presence. The levels of phospholipids are reduced to nanomolar levels.

EXAMPLE 2 Lysing White Blood Cells

The objective of this experiment is to determine the amount of time it takes for white blood cells to lyse. This allows optimization of red blood cell lysis to obtain the maximum amount of hemoglobin. In the process of isolating hemoglobin, red blood cells are lysed. At this time a specific amount of DI water may be added. The amount of time it takes for the red blood cells to lyse can be about 30 seconds. During this step any white blood cells that are remaining in the blood can lyse. When white blood cells lyse they can release protease enzymes which can have negative impact on harvested hemoglobin.

Procedure: 2000 mLs of whole blood is filtered through only the 100μm reservoir filter of the Cell Saver 5. Seven beakers are then filled with 200 mLs of blood. One beaker is designated as the control. The other six beakers are then assigned a specific time for lysing at times of 30 seconds and then 1, 2, 3, 4, and 5 minutes. The control beaker is started and 910 mLs of 0.9% saline solution is added. At time increments of 30 seconds, 1, 2, 3, 4, and 5 minutes a 10 mL sample is taken for white blood cell analysis.

For the remaining six beakers, 800 mLs of DI water is added. After 30 seconds 110 mLs of 9% saline is added to the first beaker to stop lysing. After being allowed to stir for approximately 30 seconds, a 10 mL sample is taken for white blood cell analysis. After 1 minute, 110 mLs of 9% saline is then added to the second beaker. Again after being allowed to stir for 30 seconds, a 10 mL sample is taken. For the remaining four beakers, 110 mLs of 9% saline is added at 2, 3, 4 and 5 minutes to stop lysing. After each time increment, a 10 mL sample is taken from each for white blood cell analysis.

Conclusion: As seen in FIG. 2, the lysing of the white blood cells can take place between 2 and 3 minutes. So in order to optimize red blood cell lysis, lysing can be stopped at two minutes. Due to the addition of DI water and saline, the volume is increased. The results shown in Table 7 are corrected for dilution.

TABLE 7 Control (K/MM3) Experimental (K/MM3) Time (Control Beaker) (Beakers 1-6) 30 seconds 5.9385 5.239 1 minute 5.439 4.9062 2 minutes 5.772 5.0283 3 minutes 7.3815 4.551 4 minutes 6.8265 4.03485 5 minutes 5.7165 3.3522

EXAMPLE 3 TCS Hemox-Analyzer Test

The deoxygenated, endotoxin free, stroma free, carboxymethylated cross-linked Hb (dXCMSFH) of the present invention is a stabilized tetramer of bovine hemoglobin that is locked in the tense state, so called T state. The dXCMSFH can have a p50 similar to hemoglobin within normal human red blood cells or higher. Thus, an equal amount of hemoglobin from a human red blood cell and hemoglobin from dXCMSFH, can carry the same amount of oxygen leaving the lung. However, dXCMSFH can deliver slightly more oxygen before its venous return, based upon the information obtained from the Hemox-Analyzer.

EXAMPLE 3 Rabbit Safety Trial

This rabbit trial demonstrates that dXCMSFH of the present invention can be compatible with the sensitive physiological control mechanisms mediating blood flow and pressure involving nitric oxide (NO). The dXCMSFH is infused into 25 rabbits at levels of 476 to 1143 mg/kg (average 699 mg/kg) of a 10-12% solution and results in 100 percent survival after more than 3 days. The hemoglobin without the chemical modification, when administered in 3 rabbits results in immediate death in all 3 subjects. Finally, 4 rabbits have 40-50% of their calculated blood volume removed with simultaneous dXCMSFH replacement, and subsequent daily infusion of dXCMSFH, to levels of 3871-4528 mg/kg total dose (average 4339 mg/kg). The rabbits show 100% survival and no obvious signs of morbidity. The dXCMSFH is infused into 29 rabbits without a single death due to vasoconstrictive activity. The chemical blockage of the nitric oxide (NO) binding cysteine site in dXCMSFH may be associated with the lack of vasoconstrictive activity in rabbits.

Materials: Bovine blood is collected in EDTA anticoagulant from a local slaughterhouse. A Haemonetics cell saver 5 is used to remove plasma proteins; the washed cells are passed through a leukocyte filter. Cells are lysed in 5 volumes of deionized water for 2 minutes, and then reconstituted in NS by 4 minutes. Cells and cell membranes are removed by tangential filtration on a 0.221μm filter. The stroma free hemoglobin (SFH) is then concentrated to 10-12% on a tangential membrane with a 10,000 Dalton cut off. After NO blocking with IAM, the material is crosslinked using bis-dibromosalicyl-fumarate as provided herein. After equilibration with PBS and removal of the diasprin by tangential ultrafiltration, the product is sterile filtered into IV bags and frozen at −20° C. The amount of crosslink introduced is greater than 98.5% as determined by SDS gel electrophoresis on a Beckman Coulter PA-800. The p50 for O₂ of 28-32 is determined on a TCS Hemox-Analyzer at 37° C. in pH 7.40 buffered NS. The resultant modified bovine hemoglobin at 12% is dXCMSFH.

Domestic rabbits are raised and handled locally by a 4H member. They are aged from 4-9 months of age, weighing 5-6 pounds. They include several show varieties. Rabbits are housed in separate indoor hutches, fed and watered on a daily basis. IV access is established with a 22 or 24 gauge catheter into a shaven topically anesthetized ear vein for dXCMSFH infusion. IV infusion is metered with a syringe pump; the total volume usually given over 45-60 minutes. Blood is removed from rabbits by inserting a 20-22 gauge catheter into the artery of the other ear. Procedures and infusions are done using a Velcro cloth wrap type restraint.

Methods: Protocol A: Rabbits are prepared for infusion as above. The amount of dXCMSFH to be infused is based upon 10% of the estimated blood volume of a rabbit (56 ml/kg). This amount is placed into the syringe, and infused by the pump over 45-60 minutes. The IV catheter is then removed from the rabbit's ear and the rabbit returned to its cage for observation. The control rabbits receive Hb without the NO blockage chemical modification.

Protocol B: Venous and arterial access is prepared as above. A significant amount of rabbit blood, 54-75 cc, is removed from the arterial catheter while simultaneously an identical volume of dXCMSFH is infused through the venous access. This procedure is accomplished in 12-20 minutes total time. On subsequent days 2 and 3, 15 cc of dXCMSFH is infused through the heparin locked venous catheter over a 30 minute interval, twice each day with 4 hours between infusions.

Results: Twenty-five (25) rabbits are infused according to protocol A, over an approximately 1 hour period as shown in Table 8. All of these rabbits live beyond 72 hours up to 75 days, without any deaths. Three (3) rabbits are infused with Hb without the NO blockage chemical modification. All of these rabbits die within 7-12 minutes of starting the infusion. Deaths as noted Table 9 involve strenuous activity, squealing, and rigid posturing. Four (4) rabbits receive large amounts of dXCMSFH, according to protocol B, as shown in Table 10. All of these rabbits are alive and well for more than two weeks, without any subsequent deaths.

TABLE 8 Dose Rabbit Weight dXCMSFH ID (Kg) (mg/Kg) Observations Outcome 11 2.47 567 A, C, D, E Live 15 2.4 583 A, C, D, E Live 13 2.62 534 A, C, D, E Live B7 2.36 636 A, C, D, E Live CO 2.52 476 A, C, D, E Live B3 2.24 670 A, C, D, E Live B1 2.8 714 A, C, D, E, F Live C02 2.65 566 A, C, D, E, F Live E03 2.38 630 A, C, D, E Live 04 2.1 1143 B, C, D, E Live 07 2.26 1062 B, C, D, E Live C8 2.39 1004 B, C, D, E Live MH2 3.12 808 B, C, D, E Live LHD 2.78 647 B, C, D, E Live D03 2.3 783 B, C, D, E Live D01 2.06 874 B, C, D, E Live 05 2.29 629 B, C, D, E Live 03 2.02 713 B, C, D, E Live 01 2.12 679 B, C, D, E, F Live JSD 2.13 676 B, C, D, E, F Live D04 2.26 637 B, C, D, E Live MH4 2.51 574 B, C, D, E Live K03 2.27 634 B, C, D, E Live D08 2.28 632 B, C, D, E Live D12 2.42 595 B, C, D, E Live Observation Codes Meaning A Light pink urine followed by clear urine B Clear Urine C Normal eating D Normal drinking E Normal stool F Normal mating behavior

TABLE 9 Elapsed Dose Rabbit Weight time dSFH ID (Kg) (min) (mg/Kg) Observations Outcome 06 2.04 7 98 A, B, C expired 10 2.39 12 126 A, B, C expired 09 2.42 8 83 A, B, C expired Observation Codes Meaning A Agitated Behavior B Squealing C Rigid Posturing

TABLE 10 Dose Rabbit Weight dXCMSFH dXCMSFH ID (Kg) (mg) (mg/Kg) Observations B03 3.1 12000 3871 A, C, D, E, F F02 2.79 11500 4122 A, C, D, E F01 3.02 13800 4570 A, C, D, E F05 2.65 12700 4792 A, C, D, E Observation Codes Meaning A Light pink urine followed by clear urine B Clear Urine C Normal eating D Normal drinking E Normal stool F Normal mating behavior

Discussion: The results indicate a rabbit mortality distinction between dXCMSFH and Hb without the NO blockage chemical modification. All of the rabbits receiving dXCMSFH with reduced NO affinity remain viable and without morbidity, while control subjects receiving nearly identical material but with NO affinity remaining, suffer immediate demise from what appears to be vasoconstrictive activity. Experimental subjects receive 650-7500 mg/kg without evidence of vasoconstrictive activity, while controls suffer immediate demise at levels of 125-160 mg/kg.

EXAMPLE 4 Pig Safety Trial

This experiment demonstrates a noninvasive, nonsedating, model for the determination of relevant blood pressure and flow parameters using different ultrasound modalities causing minimal disturbance to the many physiological determinants of vascular hemodynamics. The data obtained suggests that dXCMSFH does not significantly affect normal physiological control mechanisms of vascular hemodynamics in the conscious pig. A new swine model is established that measures cardiac output and systemic vascular resistance from noninvasive measurements made without restraint, anesthesia or sedation. The dXCMSFH 12% is infused into eight (8) pigs weighing 12-15 kgs over a 90 minute period through a peripheral IV established in the ear. At various times before, during and after infusion, the cardiac output is determined with Doppler Ultrasound and a blood pressure is obtained. Each pig receives 840 mg/kg of dXCMSFH. There are no deaths in the experimental group at 72 hours when the experiment is concluded. The cardiac output remains constant during the infusion, while the systemic vascular resistance increases slightly. The preliminary interpretation of this data supports the previous observation made with rabbits; dXCMSFH does not alter the normal homeostatic mechanism of blood flow and pressure mediated by NO.

Materials: Bovine blood is collected in EDTA anticoagulant from a local slaughterhouse. A Haemonetics cell saver 5 is used to remove plasma proteins; the washed cells are passed through a leukocyte filter. Cells are lysed in 5 volumes of deionized water for 2 minutes, and then reconstituted in NS for 2 minutes. Cells and cell membranes are removed by tangential filtration on a 0.22 μm filter. The stroma free hemoglobin (SFH) is then concentrated to 10-12% on a tangential membrane with a 10,000 Dalton cut off. After NO blocking, the material is crosslinked using bis-dibromosalicyl-fumarate, as previously described. After equilibration with PBS and removal of the diasprin by tangential ultrafiltration, the product is sterile filtered into IV bags and frozen at −20° C. The amount of crosslink introduced is greater than 98.5% as determined by SDS gel electrophoresis on a Beckman Coulter PA-800. The p50 for O₂ of 28-32 is determined on a TCS Hemox-Analyzer at 37° C. in pH 7.40 buffered NS. The resultant modified bovine hemoglobin at 12% is dXCMSFH.

Methods: Domestic crossbred newly weaned pigs weighing 12-15 kg are purchased from a local breeder and housed in a suitable barn. They have constant access to food and water, pen area is cleaned once or twice daily, and the lab crew spends several hours each day handling them so that they are accustomed to being held and restrained. The experiment is started with the subject pig being weighed, an IV established with a 20 gauge catheter in an ear, a 2D echocardiogram to determine cardiac structure and aortic valve diameter, and finally a blood pressure is obtained. The amount of dXCMSFH to be infused is calculated using 7 cc/kg body weight over a 90 minute period. The pigs are petted and occasionally restrained constantly throughout the infusion.

Cardiac blood flow is determined using a USCOM Doppler ultrasound device that allows the rapid calculation of many cardiac parameters, after the initial data input of the valve diameter and the blood pressure at the time of the measurement. The cardiac output and systemic vascular resistance is obtained at roughly twenty minute intervals during the infusion of dXCMSFH.

Results: Five (5) newly weaned pigs are infused with 840 mg dXCMSFH per kg body weight, an amount equal to 10% of their normal blood volume. FIGS. 7A-D represent in order cardiac output, systemic vascular resistance, systolic blood pressure and diastolic blood pressure as a function of dXCMSFH infused corrected for body weight. All of the piglets tolerate the infusion well; there are no subject deaths. A least squares method of correlating variables is used to evaluate the data, providing slope and intercept. It is readily apparent that there is great variability in the data of any parameter, regardless of the amount of dXCMSFH infused. This relates to the fact that the subjects are not sedated, nor restrained. Arousal, toileting, and handling seem to account for most of the variation, though there are significant variations between subjects' parameters even before the initiation of the experiment.

Discussion: Oxygen delivery to tissue is dependent upon blood flow. Two determinants of tissue perfusion are cardiac output and systemic vascular resistance. The cardiac output may be sufficient to maintain adequate blood pressure, and the resistance low enough to allow adequate blood flow. Local mediation of tissue perfusion can be determined by the endothelium relaxation factor expressed by nitric oxide, and a factor of systemic vascular resistance. Cardiac workload can be determined by cardiac output and blood pressure. Profound interference of normal NO homeostatic mechanisms may lead to stroke and myocardial infarction. The noninvasive model without sedation or anesthesia as provided herein can be a test of perturbations of the normal homeostatic mechanism.

The dXCMSFH may not interfere with the normal control of blood pressure and blood flow in the conscious pig. No significant change in either cardiac output or systemic vascular resistance is observed during the infusion of 840 mg/kg body weight. This amount of material can be adequate to demonstrate vasoactive interference with the NO homeostatic mechanism if the interference is present.

Prion Safety. A number of measures are taken to ensure that the Hb of the invention are prion free. Selection of suitable animals is an initial step, choosing only fat cattle which are young and less than 30 months old. A second point for prevention of contamination is scrupulous attention to avoidance of mixing brain matter into blood. The sacrificial method of the “mushroom stunner” approach is chosen to eliminate the possibility of brain matter contamination, and thus eliminate potential introduction of prion containing materials into the collected blood. Further, when the Hb is processed, the washing procedure to remove plasma proteins will also remove prions. Additionally, when the Hb is filtered through the 300,00 Da molecular weight filter, any prions can be eliminated. Lastly, the Hb of the invention is processed through the Pall filter to remove leucocytes. At this point, small formed bodies such as prions and viruses can be removed. All of these precautions operate to secure the safety of the Hb of the invention for use in human therapeutics and emergency procedures.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. 

1. A proteinaceous iron containing compound having a molecular weight of about 60,000 daltons to about 500,000 and having at least one cysteine moiety wherein said cysteine moiety includes a thiol protecting group such that said proteinaceous compound is incapable of binding nitric oxide at said cysteine site and wherein said compound is a cross-linked tetrameric hemoglobin.
 2. The proteinaceous iron containing compound of claim 1 wherein said hemoglobin is non-pyrogenic, endotoxin free, oxygen free and stroma free.
 3. The proteinaceous iron containing compound of claim 1 wherein said hemoglobin has been crosslinked with bis 3′,5′ dibromo salicyl fumarate.
 4. The proteinaceous iron containing compound of claim 1 wherein said hemoglobin has been modified by reaction with pyridoxal-5′-phosphate.
 5. The proteinaceous iron containing compound of claim 1 wherein said hemoglobin is human hemoglobin.
 6. The proteinaceous iron containing compound of claim 1 wherein said hemoglobin is bovine or porcine hemoglobin.
 7. The proteinaceous iron containing compound of claim 1 wherein said thiol protecting group is selected from the group consisting of 4-pyridylmethyl, acetylaminomethyl, alkoxyalkyl, triphenylmethyl, carboxymethyl, acetyl, benzyl, benzoyl, tert butoxycarbonyl, p-hydroxyphenacyl, p-acetoxybenzyl, p-methoxybenzyl, 2,4-dinitrophenyl, isobutoxymethyl, tetrahydropyranyl, acetamidomethyl, benzamidomethyl, bis-carboethoxyethyl, 2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, N-alkyl carbamate, and N-alkoxyalkyl carbamate.
 8. The proteinaceous iron containing compound of claim 7 wherein said thiol protecting group is a carboxymethyl group.
 9. The proteinaceous iron containing compound of claim 1 wherein said compound has increased oxygen carrying capacity compared to the oxygen carrying capacity of extracellular native human hemoglobin.
 10. The proteinaceous iron containing compound of claim 1 wherein said compound transports oxygen with a p50 of about 20 to about
 45. 11. A composition comprising said proteinaceous iron containing compound of claim 1 and a pharmaceutically acceptable carrier.
 12. A container containing a composition comprising said proteinaceous iron containing compound of claim
 1. 13. A proteinaceous iron containing compound having a molecular weight of about 60,000 daltons to about 500,000 and having at least one cysteine moiety wherein said cysteine moiety includes a thiol protecting group wherein said compound transports oxygen with a p50 of about 20 to about 45 and wherein said compound is a cross-linked tetrameric hemoglobin.
 14. The proteinaceous iron containing compound of claim 13 wherein said hemoglobin is non-pyrogenic, endotoxin free, oxygen free and stroma free.
 15. The proteinaceous iron containing compound of claim 13 wherein said hemoglobin has been crosslinked with bis 3′, 5′dibromo salicyl fumarate.
 16. The proteinaceous iron containing compound of claim 13 wherein said hemoglobin has been modified by reaction with pyridoxal-5′-phosphate.
 17. The proteinaceous iron containing compound of claim 13 wherein said hemoglobin is human hemoglobin.
 18. The proteinaceous iron containing compound of claim 13 wherein said hemoglobin is bovine or porcine hemoglobin.
 19. The proteinaceous iron containing compound of claim 13 wherein said thiol protecting group is selected from the group consisting of 4-pyridylmethyl, acetylaminomethyl, alkoxyalkyl, triphenylmethyl, carboxymethyl, acetyl, benzyl, benzoyl, tert butoxycarbonyl, p-hydroxyphenacyl, p-acetoxybenzyl, p-methoxybenzyl, 2,4-dinitrophenyl, isobutoxymethyl, tetrahydropyranyl, acetamidomethyl, benzamidomethyl, bis-carboethoxyethyl, 2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, N-alkyl carbamate, and N-alkoxyalkyl carbamate.
 20. The proteinaceous iron containing compound of claim 19 wherein said thiol protecting group is a carboxymethyl group.
 21. The proteinaceous iron containing compound of claim 13 wherein said compound is incapable of binding to nitric oxide.
 22. A composition comprising said proteinaceous iron containing compound of claim 1 and a pharmaceutically acceptable carrier.
 23. A container containing a composition comprising said proteinaceous iron containing compound of claim
 13. 